Systems, methods, and compositions for targeted nucleic acid editing

ABSTRACT

The disclosure provides for systems, methods, and compositions for targeting and editing nucleic acids. In particular, the invention provides non-naturally occurring or engineered RNA-targeting systems comprising a RNA-targeting Cas13 protein, at least one guide molecule, and at least one adenosine deaminase protein or catalytic domain thereof.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional Application Nos.62/561,669, filed Sep. 21, 2017. The entire contents of theabove-identified applications are hereby fully incorporated herein byreference.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH

This invention was made with government support under grant numberMH110049 awarded by the National Institutes of Health. The governmenthas certain rights in the invention.

REFERENCE TO AN ELECTRONIC SEQUENCE LISTING

The contents of the electronic sequence listing (“BROD-2305WP_ST25.txt”;Size is 1,409,943 bytes and it was created on Sep. 14, 2018) is hereinincorporated by reference in its entirety.

TECHNICAL FIELD

The present invention generally relates to systems, methods, andcompositions for targeting and editing nucleic acids, in particular forprogrammable deamination of adenine at a target locus of interest.

BACKGROUND

Recent advances in genome sequencing techniques and analysis methodshave significantly accelerated the ability to catalog and map geneticfactors associated with a diverse range of biological functions anddiseases. Precise genome targeting technologies are needed to enablesystematic reverse engineering of causal genetic variations by allowingselective perturbation of individual genetic elements, as well as toadvance synthetic biology, biotechnological, and medical applications.Although genome-editing techniques such as designer zinc fingers,transcription activator-like effectors (TALEs), or homing meganucleasesare available for producing targeted genome perturbations, there remainsa need for new genome engineering technologies that employ novelstrategies and molecular mechanisms and are affordable, easy to set up,scalable, and amenable to targeting multiple positions within theeukaryotic genome. This would provide a major resource for newapplications in genome engineering and biotechnology.

Programmable deamination of cytosine has been reported and may be usedfor correction of A→G and T→C point mutations. For example, Komor etal., Nature (2016) 533:420-424 reports targeted deamination of cytosineby APOBEC1 cytidine deaminase in a non-targeted DNA stranded displacedby the binding of a Cas9-guide RNA complex to a targeted DNA strand,which results in conversion of cytosine to uracil. See also Kim et al.,Nature Biotechnology (2017) 35:371-376; Shimatani et al., NatureBiotechnology (2017) doi:10.1038/nbt.3833; Zong et al., NatureBiotechnology (2017) doi:10.1038/nbt.3811; Yang Nature Communication(2016) doi:10.1038/ncomms13330.

SUMMARY

In one aspect, the present disclosure includes an engineered,non-naturally occurring system comprising a catalytically inactive Cas13effector protein (dCas13) or a nucleotide sequence encoding thecatalytically inactive Cas13 effector protein.

In some embodiments, the dCas13 protein is truncated at a C terminus, anN terminus, or both. In some embodiments, the dCas13 is truncated by atleast 20, at least 40, at least 60, at least 80, at least 100, at least120, at least 140, at least 160, at least 180, at least 200, at least220, at least 240, at least 260, or at least 300 amino acids on the Cterminus. In some embodiments, the dCas13 is truncated by at least 20,at least 40, at least 60, at least 80, at least 100, at least 120, atleast 140, at least 160, at least 180, at least 200, at least 220, atleast 240, at least 260, or at least 300 amino acids on the N terminus.In some embodiments, the truncated form of the Cas13 effector proteinhas been truncated at C-terminal Δ984-1090, C-terminal Δ1026-1090,C-terminal Δ1053-1090, C-terminal Δ934-1090, C-terminal Δ884-1090,C-terminal Δ834-1090, C-terminal Δ784-1090, or C-terminal Δ734-1090,wherein amino acid positions of the truncations correspond to amino acidpositions of Prevotella sp. P5-125 Cas13b protein. In some embodiments,the truncated form of the Cas13 effector protein has been truncated atC-terminal Δ795-1095, wherein amino acid positions of the truncationcorrespond to amino acid positions of Riemerella anatipestifer Cas13bprotein. In some embodiments, the truncated form of the Cas13 effectorprotein has been truncated at C-terminal Δ875-1175, C-terminalΔ895-1175, C-terminal Δ915-1175, C-terminal Δ935-1175, C-terminalΔ955-1175, C-terminal Δ975-1175, C-terminal Δ995-1175, C-terminalΔ1015-1175, C-terminal Δ1035-1175, C-terminal Δ1055-1175, C-terminalΔ1075-1175, C-terminal Δ1095-1175, C-terminal Δ1115-1175, C-terminalΔ1135-1175, C-terminal Δ1155-1175, wherein amino acid positionscorrespond to amino acid positions of Porphyromonas gulae Cas13bprotein. In some embodiments, the truncated form of the Cas13 effectorprotein has been truncated at N-terminal Δ1-125, N-terminal Δ1-88, orN-terminal Δ1-72, wherein amino acid positions of the truncationscorrespond to amino acid positions of Prevotella sp. P5-125 Cas13bprotein. In some embodiments, the dCas13 comprises a truncated form of aCas13 effector protein at an HEPN domain of the Cas13 effector protein.

In some embodiments, the Cas13 effector protein is Cas13a. In someembodiments, the Cas13 effector protein is Cas13b. In some embodiments,the Cas13 effector protein is Cas13c or Cas13d.

In some embodiments, the system further comprises a functional componentor wherein the nucleotide sequence further encodes the functionalcomponent. In some embodiments, the functional component is a baseediting component. In some embodiments, the base editing componentcomprises an adenosine deaminase, a cytidine deaminase, or a catalyticdomain thereof. In some embodiments, the adenosine deaminase, thecytidine deaminase, or the catalytic domain thereof, is fused to thedCas13. In some embodiments, the adenosine deaminase, the cytidinedeaminase, or the catalytic domain thereof, is fused to the dCas13 by alinker. In some embodiments, the adenosine deaminase, the cytidinedeaminase, or the catalytic domain thereof, is inserted into an internalloop of the dCas13. In some embodiments, the adenosine deaminase, thecytidine deaminase, or the catalytic domain thereof, is linked to anadaptor protein. In some embodiments, the adaptor protein is selectedfrom MS2, PP7, Qβ, F2, GA, fr, JP501, M12, R17, BZ13, JP34, JP500, KU1,M11, MX1, TW18, VK, SP, FI, ID2, NL95, TW19, AP205, ϕCb5, ϕCb8r, ϕCb12r,ϕCb23r, 7s and PRR1. In some embodiments, the adenosine deaminase, thecytidine deaminase, or the catalytic domain thereof, is a human,cephalopod, or Drosophila protein.

In some embodiments, the dCas13 is a split Cas13 effector protein. Insome embodiments, the split Cas13 effector protein is a first split Cas13 effector protein and is capable of fusing to a second split Cas13effector protein to form a catalytically active Cas13 effector protein.In some embodiments, fusing of the first and the second Cas13 effectorproteins is inducible.

In some embodiments, the functional component is a transcription factoror an active domain thereof. In some embodiments, the dCas13 is fusedwith the transcription factor or the active domain thereof. In someembodiments, the system further comprises a guide sequence.

In another aspect, the present disclosure includes a vector systemcomprising one or more vectors encoding the dCas13 of disclosed herein.

In another aspect, the present disclosure includes an in vitro or exvivo host cell or progeny thereof or cell line or progeny thereofcomprising the system disclosed herein.

In another aspect, the present disclosure includes a method forprogrammable and targeted base editing of a target sequence comprisingdelivering of the system disclosed herein to a cell. In someembodiments, the method further comprises determining the targetsequence of interest and selecting an adenosine deaminase, cytidinedeaminase, or catalytic domain thereof which most efficiently deaminatesan adenine or cytidine present in the target sequence. In someembodiments, the deamination remedies a disease caused by transcriptscontaining a pathogenic T(U)→C, A→G, G→A, or C→T point mutation.

In another aspect, the present disclosure includes a method formodifying a nucleic acid at a target locus, the method comprisingdelivering (1) the system of claim 1, wherein the dCas13 is a firstsplit Cas13 effector protein, and (2) a second split Cas13 effectorprotein, wherein the first and the second split Cas13 effector proteinsare capable of forming a catalytically active Cas13 effector protein

In another aspect, the present disclosure includes a method forregulating a target gene in a cell, the method comprising delivering thesystem disclosed herein, wherein the dCas13 is fused to a transcriptionfactor or an active domain thereof.

These and other aspects, objects, features, and advantages of theexample embodiments will become apparent to those having ordinary skillin the art upon consideration of the following detailed description ofillustrated example embodiments.

BRIEF DESCRIPTION OF THE DRAWINGS

An understanding of the features and advantages of the present inventionwill be obtained by reference to the following detailed description thatsets forth illustrative embodiments, in which the principles of theinvention may be utilized, and the accompanying drawings of which:

FIG. 1 illustrates an example embodiment of the invention for targeteddeamination of adenine at a target RNA sequence of interest, exemplifiedherein with a Cas13b protein.

FIG. 2 illustrates the Development of RNA editing as a therapeuticstrategy to treat human disease at the transcript level such as whenusing Cas13b. Schematic of RNA base editing by Cas13-ADAR2 fusiontargeting an engineered pre-termination stop codon in the luciferasetranscript.

FIG. 3 Guide position and length optimization to restore luciferaseexpression.

FIG. 4 Exemplary sequences of adenine deaminase proteins. (SEQ ID NOs:650-656)

FIG. 5 Guides used in an exemplary embodiment (SEQ ID NOs: 657-659 and762)

FIG. 6: Editing efficiency correlates to edited base being further awayfrom the DR and having a long RNA duplex, which is accomplished byextending the guide length

FIG. 7 Greater editing efficiency the further the editing site is awayfrom the DR/protein binding area.

FIG. 8 Distance of edited site from DR.

FIGS. 9A and 9B: Fused ADAR1 or ADAR2 to Cas13b12 (double R HEPN mutant)on the N or C-terminus. Guides are perfect matches to the stop codon inluciferase. Signal appears correlated with distance between edited baseand 5′ end of the guide, with shorter distances providing betterediting.

FIG. 10: Cluc/Gluc tiling for Cas13a/Cas13b interference

FIGS. 11A-11C: ADAR editing quantification by NGS (luciferase reporter).

FIG. 12: ADAR editing quantification by NGS (KRAS and PPIB).

FIGS. 13A-13C: Cas13a/b+shRNA specificity from RNA Seq.

FIG. 14: Mismatch specificity to reduce off targets (A:A or A:G) (SEQ IDNOs: 661-668)

FIG. 15: Mismatch for on-target activity.

FIG. 16: ADAR Motif preference.

FIG. 17: Larger bubbles to enhance RNA editing efficiency.

FIG. 18: Editing of multiple A's in a transcript (SEQ ID NOs: 669-672).

FIG. 19: Guide length titration for RNA editing.

FIGS. 20A-20F: Mammalian codon-optimized Cas13b orthologs mediate highlyefficient RNA knockdown. (FIG. 20A) Schematic of representative Cas13a,Cas13b, and Cas13c loci and associated crRNAs. (FIG. 20B) Schematic ofluciferase assay to measure Cas13a cleavage activity in HEK293FT cells.(FIG. 20C) RNA knockdown efficiency using two different guides targetingCluc with 19 Cas13a, 15 Cas13b, and 5 Cas13c orthologs. Luciferaseexpression is normalized to the expression in non-targeting guidecontrol conditions. (FIG. 20D) The top 7 orthologs performing in part Care assayed for activity with three different NLS and NES tags with twodifferent guide RNAs targeting Cluc. (FIG. 20E) Cas13b12 and Cas13a2(LwCas13a) are compared for knockdown activity against Gluc and Cluc.Guides are tiled along the transcripts and guides between Cas13b12 andCas13a2 are position matched. (FIG. 20F) Guide knockdown for Cas13a2,Cas13b6, Cas13b11, and Cas13b12 against the endogenous KRAS transcriptand are compared against corresponding shRNAs.

FIGS. 21A-21G: Cas13 enzymes mediate specific RNA knockdown in mammaliancells. (FIG. 21A) Schematic of semi-degenerate target sequences forCas13a/b mismatch specificity testing (SEQ ID NOs: 673-294). (FIG. 21B)Heatmap of single mismatch knockdown data for Cas13 a/b. Knockdown isnormalized to non-targeting (NT) guides for each enzyme. (FIG. 21C)Double mismatch knockdown data for Cas13a. The position of each mismatchis indicated on the X and Y axes. Knockdown data is the sum of alldouble mismatches for a given set of positions. Data is normalized to NTguides for each enzyme. (FIG. 21D) Double mismatch knockdown data forCas13b. See C for description. (FIG. 21E) RNA-seq data comparingtranscriptome-wide specificity for Cas13 a/b and shRNA forposition-matched guides. The Y axis represents read counts for thetargeting condition and the X axis represents counts for thenon-targeting condition. (FIG. 21F) RNA expression as calculated fromRNA-seq data for Cas13 a/b and shRNA. (FIG. 21G) Significant off-targetsfor Cas13 a/b and shRNA from RNA-seq data. Significant off-targets werecalculated using FDR<0.05.

FIGS. 22A-22F: Catalytically inactive Cas13b-ADAR fusions enabletargeted RNA editing in mammalian cells. (FIG. 22A) Schematic of RNAediting with Cas13b-ADAR fusion proteins to remove stop codons on theCypridina luciferase transcript. (FIG. 22B) RNA editing comparisonbetween Cas13b fused with wild-type ADAR2 and Cas13b fused with thehyperactive ADAR2 E488Q mutant for multiple guide positions. Luciferaseexpression is normalized to Gaussia luciferase control values. (FIG.22C) RNA editing comparisons between 30, 50, 70, and 84 nt guidesdesigned to target various positions surrounding the editing site. (FIG.22D) Schematic showing the position and length of guides used forsequencing quantification relative to the stop codon on the Cypridinaluciferase transcript (SEQ ID NO: 695). (FIG. 22E) On- and off-targetediting efficiencies for each guide design at the corresponding adeninebases on the Cypridina luciferase transcript as quantified bysequencing. (FIG. 22F) Luciferase readout of guides with varied basesopposite to the targeted adenine.

FIG. 23: Endogenous RNA editing with Cas13b-ADAR fusions. Nextgeneration sequencing of endogenous Cas13b12-ADAR editing of endogenousKRAS and PPIB loci. Two different regions per transcript were targetedand A→G editing was quantified at all adenines in the vicinity of thetargeted adenine.

FIG. 24: Strategy for determining optimal guide position.

FIGS. 25A-25C: (FIG. 25A) Cas13b-huADAR2 promotes repair of mutatedluciferase transcripts. (FIG. 25B) Cas13b-huADAR1 promotes repair ofmutated luciferase transcripts. (FIG. 25C) Comparison of human ADAR1 andhuman ADAR2.

FIG. 26: Comparison of E488Q vs. wt dADAR2 editing. E488Q is ahyperactive mutant of dADAR2.

FIGS. 27A-27B: Transcripts targeted by Cas13b-huADAR2-E488Q contain theexpected A-G edit. (FIG. 27A) heatmap. (FIG. 27B) Positions in template.Only A sites are shown with the editing rate to G as in heatmap.

FIGS. 28A-28B: Endogenous tiling of guides. (FIG. 28A) KRAS: heatmap.(top) Positions in template (bottom). Only A sites are shown with theediting rate to G as in heatmap. (FIG. 28B) PPIB: heatmap. (top)Positions in template (bottom). Only A sites are shown with the editingrate to G as in heatmap.

FIG. 29: Non-targeting editing.

FIG. 30: Linker optimization.

FIG. 31: Cas13b ADAR can be used to correct pathogenic A>G mutationsfrom patients in expressed cDNAs.

FIG. 32: Cas13b-ADAR has a slight restriction on 5′ G motifs.

FIG. 33: Screening degenerate PFS locations for effect on editingefficiency. All PFS (4-N) identities have higher editing thannon-targeting (SEQ ID NOs: 696-699).

FIG. 34: Reducing off-target editing in the target transcript.

FIG. 35: Reducing off-target editing in the target transcript.

FIGS. 36A-36B: Cas13b-ADAR transcriptome specificity. On-target editingis 71%. (FIG. 36A) targeting guide; 482 significant sites. (FIG. 36B)non-targeting guide; 949 significant sites. Note that chromosome 0 isGluc and chromosome 1 is Cluc; human chromosomes are then in order afterthat.

FIGS. 37A-37D: Cas13b-ADAR transcriptome specificity. (FIGS. 37A and37B) targeting guide. (FIGS. 37C and 37D) non-targeting guide.

FIG. 38: Cas13b has the highest efficiency compared to competing ADARediting strategies.

FIGS. 39A-39D: Competing RNA editing systems. (FIGS. 39A-39B) BoxB;on-target editing is 63%; (FIG. 39A) targeting guide—2020 significantsites; (FIG. 39B) non-targeting guide—1805 significant sites. (FIGS.39C-39D) Stafforst; on-target editing is 36%; (FIG. 39C) targetingguide—176 significant sites; (FIG. 39D) non-targeting guide—186significant sites.

FIG. 40: Dose titration of ADAR. crRNA amount is constant.

FIGS. 41A-41D: Dose response effect on specificity. (FIGS. 41A-41B) 150ng Cas13-ADAR; on-target editing is 83%; (FIG. 41A) targeting guide—1231significant sites; (FIG. 41B) non-targeting guide—520 significant sites.(FIGS. 41C-41D) 10 ng Cas13-ADAR; on-target editing is 80%; (FIG. 41C)targeting guide—347 significant sites; (FIG. 41D) non-targetingguide—223 significant sites.

FIGS. 42A-42B: ADAR1 seems more specific than ADAR2. On-target editingis 29%. (FIG. 42A) targeting guide; 11 significant sites. (FIG. 42B)non-targeting guide; 6 significant sites. Note that chromosome 0 is Glucand chromosome 1 is Cluc; human chromosomes are then in order afterthat.

FIGS. 43A-43D: ADAR specificity mutants have enhanced specificity. (FIG.43A) Targeting guide. (FIG. 43B) Non-targeting guide. (FIG. 43C)Targeting to non-targeting ratio. (FIG. 43D) Targeting and non-targetingguide.

FIG. 44: ADAR mutant luciferase results plotted along the contact pointsof each residue with the RNA target.

FIG. 45: ADAR specificity mutants have enhanced specificity. Purplepoints are mutants selected for whole transcriptome off-target NGSanalysis. Red point is the starting point (i.e. E488Q mutant). Note thatall additional mutants also have the E488Q mutation.

FIGS. 46A-46B: ADAR mutants are more specific according to NGS. (FIG.46A) on target. (FIG. 46B) Off-target.

FIGS. 47A-47B: Luciferase data on ADAR specificity mutants matches theNGS. (FIG. 47A) Targeting guide selected for NGS. (FIG. 47B)Non-targeting guide selected for NGS. Luciferase data matches the NGSdata in FIGS. 46A-46B. The orthologs that have fewer activity withnon-targeting guide have fewer off-targets across the transcriptome andtheir on-target editing efficiency can be predicted by the targetingguide luciferase condition.

FIG. 48: C-terminal truncations of Cas13b 12 are still highly active inADAR editing.

FIGS. 49A-49G: Characterization of a highly active Cas13b ortholog forRNA knockdown FIG. 49A) Schematic of stereotypical Cas13 loci andcorresponding crRNA structure. FIG. 49B) Evaluation of 19 Cas13a, 15Cas13b, and 7 Cas13c orthologs for luciferase knockdown using twodifferent guides. Orthologs with efficient knockdown using both guidesare labeled with their host organism name. FIG. 49C) PspCas13b andLwaCas13a knockdown activity are compared by tiling guides against Glucand measuring luciferase expression. FIG. 49D) PspCas13b and LwaCas13aknockdown activity are compared by tiling guides against Cluc andmeasuring luciferase expression. FIG. 49E) Expression levels inlog2(transcripts per million (TPM)) values of all genes detected inRNA-seq libraries of non-targeting control (x-axis) compared toGluc-targeting condition (y-axis) for LwaCas13a (red) and shRNA (black).Shown is the mean of three biological replicates. The Gluc transcriptdata point is labeled. FIG. 49F) Expression levels in log2(transcriptsper million (TPM)) values of all genes detected in RNA-seq libraries ofnon-targeting control (x-axis) compared to Gluc-targeting condition(y-axis) for PspCas13b (blue) and shRNA (black). Shown is the mean ofthree biological replicates. The Gluc transcript data point is labeled.FIG. 49G) Number of significant off-targets from Gluc knockdown forLwaCas13a, PspCas13b, and shRNA from the transcriptome wide analysis inFIG. 49E and FIG. 49F.

FIGS. 50A-50E: Engineering dCas13b-ADAR fusions for RNA editing FIG.50A) Schematic of RNA editing by dCas13b-ADAR fusion proteins. FIG. 50B)Schematic of Cypridina luciferase W85X target and targeting guide design(SEQ ID NOs: 700 and 701). FIG. 50C) Quantification of luciferaseactivity restoration for Cas13b-dADAR1 (left) and Cas13b-ADAR2-cd(right) with tiling guides of length 30, 50, 70, or 84 nt. FIG. 50D)Schematic of target site for targeting Cypridinia luciferase W85X. FIG.50E) Sequencing quantification of A→I editing for 50 nt guides targetingCypridinia luciferase W85X (SEQ ID NO: 702).

FIGS. 51A-51D: FIG. 51A Measuring sequence flexibility for RNA editingby REPAIRv1 Schematic of screen for determining Protospacer FlankingSite (PFS) preferences of RNA editing by REPAIRv1. (SEQ ID NO: 703).FIG. 51B) Distributions of RNA editing efficiencies for all 4-N PFScombinations at two different editing sites. FIG. 51C) Quantification ofthe percent editing of REPAIRv1 at Cluc W85 across all possible 3 basemotifs (SEQ ID NO: 704). FIG. 51D) Heatmap of 5′ and 3′ base preferencesof RNA editing at Cluc W85 for all possible 3 base motifs

FIGS. 52A-52G: Correction of disease-relevant mutations with REPAIRv1FIG. 52A) Schematic of target and guide design for targeting AVPR2878G>A (SEQ ID NO: 705-708). FIG. 52B) The 878G>A mutation in AVPR2 iscorrected to varying percentages using REPAIRv1 with three differentguide designs. FIG. 52C) Schematic of target and guide design fortargeting FANCC 1517G>A. (SEQ ID NO: 709-712). FIG. 52D) The 1517G>Amutation in FANCC is corrected to varying percentages using REPAIRv1with three different guide designs. FIG. 52E) Quantification of thepercent editing of 34 different disease-relevant G>A mutations usingREPAIRv1. FIGS. 52F-52G) Analysis of all the possible G>A mutations thatcould be corrected as annotated by the ClinVar database. Thedistribution of editing motifs for all G>A mutations in ClinVar is shownversus the editing efficiency by REPAIRv1 per motif as quantified on theGluc transcript.

FIGS. 53A-53D: Characterizing specificity of REPAIRv1 FIG. 53A)Schematic of KRAS target site and guide design. (SEQ ID NOs: 713-720).FIG. 53B) Quantification of percent editing for tiled KRAS-targetingguides. Editing percentages are shown at the on-target and neighboringadenosine sites. For each guide, the region of duplex RNA is indicatedby a red rectangle. FIG. 53C) Transcriptome-wide sites of significantRNA editing by REPAIRv1 with Cluc targeting guide. The on-target siteCluc site (254 A>G) is highlighted in orange. FIG. 53D)Transcriptome-wide sites of significant RNA editing by REPAIRv1 withnon-targeting guide.

FIGS. 54A-54F: Rational mutagenesis of ADAR2 to improve the specificityof REPAIRv1 FIG. 54A) Quantification of luciferase signal restoration byvarious dCas13-ADAR2 mutants as well as their specificity score plottedalong a schematic for the contacts between key ADAR2 deaminase residuesand the dsRNA target. The specificity score is defined as the ratio ofthe luciferase signal between targeting guide and non-targeting guideconditions. FIG. 54B) Quantification of luciferase signal restoration byvarious dCas13-ADAR2 mutants versus their specificity score. FIG. 54C)Measurement of the on-target editing fraction as well as the number ofsignificant off-targets for each dCas13-ADAR2 mutant by transcriptomewide sequencing of mRNAs. FIG. 54D) Transcriptome-wide sites ofsignificant RNA editing by REPAIRv1 and REPAIRv2 with a guide targetinga pretermination site in Cluc. The on-target Cluc site (254 A>G) ishighlighted in orange. FIG. 54E) RNA sequencing reads surrounding theon-target Cluc editing site (254 A>G) highlighting the differences inoff-target editing between REPAIRv1 and REPAIRv2. All A>G edits arehighlighted in red while sequencing errors are highlighted in blue (SEQID NO: 721). FIG. 54F) RNA editing by REPAIRv1 and REPAIRv2 with guidestargeting an out-of-frame UAG site in the endogenous KRAS and PPIBtranscripts. The on-target editing fraction is shown as a sideways barchart on the right for each condition row. The duplex region formed bythe guide RNA is shown by a red outline box.

FIGS. 55A-55C: Bacterial screening of Cas13b orthologs for in vivoefficiency and PFS determination. FIG. 55A) Schematic of bacterial assayfor determining the PFS of Cas13b orthologs. Cas13b orthologs withbeta-lactamase targeting spacers are co-transformed with beta-lactamaseexpression plasmids and subjected to double selection. (SEQ ID NO: 722).FIG. 55B) Quantitation of interference activity of Cas13b orthologstargeting beta-lactamase as measured by colony forming units (cfu). FIG.55C) PFS logos for Cas13b orthologs as determined by depleted sequencesfrom the bacterial assay.

FIGS. 56A-56E: Optimization of Cas13b knockdown and furthercharacterization of mismatch specificity. FIG. 56A) Gluc knockdown withtwo different guides is measured using the top 2 Cas13a and top 4 Cas13borthologs fused to a variety of nuclear localization and nuclear exporttags. FIG. 56B) Knockdown of KRAS is measured for LwaCas13a, RanCas13b,PguCas13b, and PspCas13b with four different guides and compared to fourposition-matched shRNA controls. FIG. 56C) Schematic of the single anddouble mismatch plasmid libraries used for evaluating the specificity ofLwaCas13a and PspCas13b knockdown. Every possible single and doublemismatch is present in the target sequence as well as in 3 positionsdirectly flanking the 5′ and 3′ ends of the target site. (SEQ ID NO:723-735). FIG. 56D) The depletion level of transcripts with theindicated single mismatches are plotted as a heatmap for both theLwaCas13a and PspCas13b conditions. FIG. 56E) The depletion level oftranscripts with the indicated double mismatches are plotted as aheatmap for both the LwaCas13a and PspCas13b conditions (SEQ ID NO:736).

FIGS. 57A-57F: Characterization of design parameters for dCas13-ADAR2RNA editing FIG. 57A) Knockdown efficiency of Gluc targeting forwildtype Cas13b and catalytically inactive H133A/H1058A Cas13b(dCas13b). FIG. 57B) Quantification of luciferase activity restorationby dCas13b fused to either the wildtype ADAR2 catalytic domain or thehyperactive E488Q mutant ADAR2 catalytic catalytic domain, tested withtiling Cluc targeting guides. FIG. 57C) Guide design and sequencingquantification of A→I editing for 30 nt guides targeting Cypridinialuciferase W85X (SEQ ID NOs: 737-745). FIG. 57D) Guide design andsequencing quantification of A→I editing for 50 nt guides targeting PPIB(SEQ ID NO: 746-753). FIG. 57E) Influence of linker choice on luciferaseactivity restoration by REPAIRv1. FIG. 57F) Influence of base identifyopposite the targeted adenosine on luciferase activity restoration byREPAIRv1. (SEQ ID NO: 754-755).

FIG. 58: ClinVar motif distribution for G>A mutations. The number ofeach possible triplet motif observed in the ClinVar database for all G>Amutations.

FIG. 59: Truncations of dCas13b still have functional RNA editing.Various N-terminal and C-terminal truncations of dCas13b allow for RNAediting as measured by restoration of luciferase signal.

FIGS. 60A-60F: Comparison of other programmable ADAR systems with thedCas13-ADAR2 editor. FIG. 60A) Schematic of two programmable ADARschemes: BoxB-based targeting and full length ADAR2 targeting. In theBoxB scheme (top), the ADAR2 deaminase domain (ADAR2_(DD)(E488Q)) isfused to a small bacterial virus protein called lambda N (λN), whichbinds specifically a small RNA sequence called BoxB-λ. A guide RNAcontaining two BoxB-λ hairpins can then guide the ADAR2_(DD)(E488Q), -λNfor site specific editing. In the full length ADAR2 scheme (bottom), thedsRNA binding domains of ADAR2 bind a hairpin in the guide RNA, allowingfor programmable ADAR2 editing. (SEQ ID NOs: 756-760). FIG. 60B)Transcriptome-wide sites of significant RNA editing byBoxB-ADAR2_(DD)(E488Q) with a guide targeting Cluc and a non-targetingguide. The on-target Cluc site (254 A>G) is highlighted in orange. FIG.60C) Transcriptome-wide sites of significant RNA editing by ADAR2 with aguide targeting Cluc and a non-targeting guide. The on-target Cluc site(254 A>G) is highlighted in orange. FIG. 60D) Transcriptome-wide sitesof significant RNA editing by REPAIRv1 with a guide targeting Cluc and anon-targeting guide. The on-target Cluc site (254 A>G) is highlighted inorange. FIG. 60E) Quantitation of on-target editing rate percentage forBoxB-ADAR2_(DD)(E488Q), ADAR2, and REPAIRv1 for targeting guides againstCluc. FIG. 60F) Overlap of off-target sites between different targetingand non-targeting conditions for programmable ADAR systems.

FIGS. 61A-61C: Efficiency and specificity of dCas13b-ADAR2 mutants FIG.61A) Quantitation of luciferase activity restoration bydCas13b-ADAR2_(DD)(E488Q) mutants for Cluc-targeting and non-targetingguides. FIG. 61B) Relationship between the ratio of targeting andnon-targeting guides and the number of RNA-editing off-targets asquantified by transcriptome-wide sequencing FIG. 61C) Quantification ofnumber of transcriptome-wide off-target RNA editing sites versuson-target Cluc editing efficiency for dCas13b-ADAR2_(DD)(E488Q) mutants.

FIGS. 62A-62B: Transcriptome-wide specificity of RNA editing bydCas13b-ADAR2_(DD)(E488Q) mutants FIG. 62A) Transcriptome-wide sites ofsignificant RNA editing by dCas13b-ADAR2_(DD)(E488Q) mutants with aguide targeting Cluc. The on-target Cluc site (254 A>G) is highlightedin orange. FIG. 62B) Transcriptome-wide sites of significant RNA editingby dCas13b-ADAR2_(DD)(E488Q) mutants with a non-targeting guide.

FIGS. 63A-63C: Characterization of motif biases in the off-targets ofdCas13b-ADAR2_(DD)(E488Q) editing. FIG. 63A) For eachdCas13b-ADAR2_(DD)(E488Q) mutant, the motif present across all A>Goff-target edits in the transcriptome is shown. FIG. 63B) Thedistribution of off-target A>G edits per motif identity is shown forREPAIRv1 with targeting and non-targeting guide. FIG. 63C) Thedistribution of off-target A>G edits per motif identity is shown forREPAIRv2 with targeting and non-targeting guide.

FIGS. 64A-63F: Further characterization of REPAIRv1 and REPAIRv2off-targets. FIG. 64A) Histogram of the number of off-targets pertranscript for REPAIRv1. FIG. 64B) Histogram of the number ofoff-targets per transcript for REPAIRv2. FIG. 64C) Variant effectprediction of REPAIRv1 off targets. FIG. 64D) Distribution of potentialoncogenic effects of REPAIRv1 off targets. FIG. 64E) Variant effectprediction of REPAIRv2 off targets. FIG. 64F) Distribution of potentialoncogenic effects of REPAIRv2 off targets.

FIGS. 65A-65C: RNA editing efficiency and specificity of REPAIRv1 andREPAIRv2. FIG. 65A) Quantification of percent editing of KRAS withKRAS-targeting guide 1 at the targeted adenosine and neighboring sitesfor REPAIRv1 and REPAIRv2. FIG. 65B) Quantification of percent editingof KRAS with KRAS-targeting guide 3 at the targeted adenosine andneighboring sites for REPAIRv1 and REPAIRv2. FIG. 65C) Quantification ofpercent editing of PPIB with PPIB-targeting guide 2 at the targetedadenosine and neighboring sites for REPAIRv1 and REPAIRv2.

FIGS. 66A-66G: Demonstration of all potential codon changes with a A>GRNA editor. FIG. 66A) Table of all potential codon transitions enabledby A>I editing. FIGS. 66B-66G) A codon table demonstrating all thepotential codon transitions enabled by A>I editing.

FIG. 67 shows the test results of RNA editing activities on Cas13b6.

FIG. 68 shows the test results of RNA editing activities on Cas13b11.

FIG. 69 shows the test results of RNA editing activities on Cas13b12.

FIG. 70 shows a schematic of dCas13b6(Δ795-1095)-REPAIR and the editingrate of the construct.

FIG. 71 shows modulating editing at a site in the potassium ion channelKcna1 that is already a natural ADAR2 substrate by delivering thedCas13b6(Δ795-1095)-REPAIR.

FIG. 72 shows a schematic of schematic of thedCas13b6(Δ795-1095)-GS-HIVNES-GS-huADAR2dd.

FIG. 73 REPAIR assay of pgCas13b C-terminal truncations.

FIG. 74 shows alignment tree of ADAR orthologs that can be used with thesystems and methods herein.

The figures herein are for illustrative purposes only and are notnecessarily drawn to scale.

DETAILED DESCRIPTION OF THE EXAMPLE EMBODIMENTS General Definitions

Unless defined otherwise, technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this disclosure pertains. Definitions of common termsand techniques in molecular biology may be found in Molecular Cloning: ALaboratory Manual, 2^(nd) edition (1989) (Sambrook, Fritsch, andManiatis); Molecular Cloning: A Laboratory Manual, 4^(th) edition (2012)(Green and Sambrook); Current Protocols in Molecular Biology (1987) (F.M. Ausubel et al. eds.); the series Methods in Enzymology (AcademicPress, Inc.): PCR 2: A Practical Approach (1995) (M. J. MacPherson, B.D. Hames, and G. R. Taylor eds.): Antibodies, A Laboraotry Manual (1988)(Harlow and Lane, eds.): Antibodies A Laboraotry Manual, 2^(nd) edition2013 (E. A. Greenfield ed.); Animal Cell Culture (1987) (R. I. Freshney,ed.); Benjamin Lewin, Genes IX, published by Jones and Bartlet, 2008(ISBN 0763752223); Kendrew et al. (eds.), The Encyclopedia of MolecularBiology, published by Blackwell Science Ltd., 1994 (ISBN 0632021829);Robert A. Meyers (ed.), Molecular Biology and Biotechnology: aComprehensive Desk Reference, published by VCH Publishers, Inc., 1995(ISBN 9780471185710); Singleton et al., Dictionary of Microbiology andMolecular Biology 2nd ed., J. Wiley & Sons (New York, N.Y. 1994), March,Advanced Organic Chemistry Reactions, Mechanisms and Structure 4th ed.,John Wiley & Sons (New York, N.Y. 1992); and Marten H. Hofker and Janvan Deursen, Transgenic Mouse Methods and Protocols, 2nd edition (2011).

Reference is made to U.S. Provisional 62/351,662 and 62/351,803, filedon Jun. 17, 2016, U.S. Provisional 62/376,377, filed on Aug. 17, 2016,U.S. Provisional 62/410,366, filed Oct. 19, 2016, U.S. Provisional62/432,240, filed Dec. 9, 2016, U.S. provisional 62/471,792 filed Mar.15, 2017, and U.S. Provisional 62/484,786 filed Apr. 12, 2017. Referenceis made to International PCT application PCT/US2017/038154, filed Jun.19, 2017. Reference is made to U.S. Provisional 62/471,710, filed Mar.15, 2017 (entitled, “Novel Cas13B Orthologues CRISPR Enzymes andSystems,” Attorney Ref: BI-10157 VP 47627.04.2149). Reference is furthermade to U.S. Provisional 62/432,553, filed Dec. 9, 2016, U.S.Provisional 62/456,645, filed Feb. 8, 2017, and U.S. Provisional62/471,930, filed Mar. 15, 2017 (entitled “CRISPR Effector System BasedDiagnostics,” Attorney Ref. BI-10121 BROD 0842P) and US Provisional ToBe Assigned, filed Apr. 12, 2017 (entitled “CRISPR Effector System BasedDiagnostics,” Attorney Ref. BI-10121 BROD 0843P)

As used herein, the singular forms “a”, “an”, and “the” include bothsingular and plural referents unless the context clearly dictatesotherwise.

The term “optional” or “optionally” means that the subsequent describedevent, circumstance or substituent may or may not occur, and that thedescription includes instances where the event or circumstance occursand instances where it does not.

The recitation of numerical ranges by endpoints includes all numbers andfractions subsumed within the respective ranges, as well as the recitedendpoints.

The terms “about” or “approximately” as used herein when referring to ameasurable value such as a parameter, an amount, a temporal duration,and the like, are meant to encompass variations of and from thespecified value, such as variations of +/−10% or less, +/−5% or less,+/−1% or less, and +/−0.1% or less of and from the specified value,insofar such variations are appropriate to perform in the disclosedinvention. It is to be understood that the value to which the modifier“about” or “approximately” refers is itself also specifically, andpreferably, disclosed.

As used herein, a “biological sample” may contain whole cells and/orlive cells and/or cell debris. The biological sample may contain (or bederived from) a “bodily fluid”. The present invention encompassesembodiments wherein the bodily fluid is selected from amniotic fluid,aqueous humour, vitreous humour, bile, blood serum, breast milk,cerebrospinal fluid, cerumen (earwax), chyle, chyme, endolymph,perilymph, exudates, feces, female ejaculate, gastric acid, gastricjuice, lymph, mucus (including nasal drainage and phlegm), pericardialfluid, peritoneal fluid, pleural fluid, pus, rheum, saliva, sebum (skinoil), semen, sputum, synovial fluid, sweat, tears, urine, vaginalsecretion, vomit and mixtures of one or more thereof. Biological samplesinclude cell cultures, bodily fluids, cell cultures from bodily fluids.Bodily fluids may be obtained from a mammal organism, for example bypuncture, or other collecting or sampling procedures.

The terms “subject,” “individual,” and “patient” are usedinterchangeably herein to refer to a vertebrate, preferably a mammal,more preferably a human. Mammals include, but are not limited to,murines, simians, humans, farm animals, sport animals, and pets.Tissues, cells and their progeny of a biological entity obtained in vivoor cultured in vitro are also encompassed.

Reference throughout this specification to “one embodiment”, “anembodiment,” “an example embodiment,” means that a particular feature,structure or characteristic described in connection with the embodimentis included in at least one embodiment of the present invention. Thus,appearances of the phrases “in one embodiment,” “in an embodiment,” or“an example embodiment” in various places throughout this specificationare not necessarily all referring to the same embodiment, but may.Furthermore, the particular features, structures or characteristics maybe combined in any suitable manner, as would be apparent to a personskilled in the art from this disclosure, in one or more embodiments.Furthermore, while some embodiments described herein include some butnot other features included in other embodiments, combinations offeatures of different embodiments are meant to be within the scope ofthe invention. For example, in the appended claims, any of the claimedembodiments can be used in any combination.

C2c2 is now known as Cas13a. It will be understood that the term “C2c2”herein is used interchangeably with “Cas13a”. As used herein, Cas13 mayrefer to Cas13a or Cas13b or Cas13c or Cas13d or other member in theCas13 family. In some examples, Cas13 may be Cas13a. In some examples,Cas13 may be Cas13b. In some examples, Cas13 may be Cas13c. In someexamples, Cas13 may be Cas13d.

All publications, published patent documents, and patent applicationscited herein are hereby incorporated by reference to the same extent asthough each individual publication, published patent document, or patentapplication was specifically and individually indicated as beingincorporated by reference.

Various embodiments are described hereinafter. It should be noted thatthe specific embodiments are not intended as an exhaustive descriptionor as a limitation to the broader aspects discussed herein. One aspectdescribed in conjunction with a particular embodiment is not necessarilylimited to that embodiment and can be practiced with any otherembodiment(s).

Overview

The embodiments disclosed herein provide systems, constructs, andmethods for using catalytically inactive Cas effector proteins (e.g.,Cas13) for various applications. In general, the systems disclosedherein may comprise a catalytically inactive Cas effector protein (e.g.,dCas13). The catalytically inactive Cas13 (dCas13) may includetruncations of Cas13 effector proteins, e.g., at the C-terminus, theN-terminus, or both. In some embodiments, the systems comprise dCas13.The dCas13 may be a catalytically inactive form of any Cas13 subtypeprotein. For example, dCas13 may be dCas13a, dCas13b, dCas13c, ordCas13d. In some examples, the dCas13 may be modified Cas13 effectorproteins from Prevotella sp. P5-125, Riemerella anatipestifer, orPorphyromonas gulae.

The systems may further comprise one or more functional components fordesired applications. In some examples, the systems may comprise a baseediting component, e.g., an adenosine deaminase or a cytidine deaminase,or a catalytic domain thereof. In some examples, the systems maycomprise a transcription factor or an active domain thereof. In thesecases, the catalytically inactive Cas effector protein may be used fordelivering the transcription factor to a target sequence. In someexamples, the catalytically inactive Cas effector protein may be a splitCas effector protein. The split Cas may be used with another split Casfor various applications. For example, the two split Cas effectorproteins may be fused to form a catalytically active Cas effectorprotein. The fusing may be controllable, e.g., using an inducing agent.

Type-V CRISPR-Cas Protein

The application describes methods using Type-V CRISPR-Cas proteins. Thisis exemplified herein with Cas13, whereby a number of orthologs orhomologs have been identified. It will be apparent to the skilled personthat further orthologs or homologs can be identified and that any of thefunctionalities described herein may be engineered into other orthologs,including chimeric enzymes comprising fragments from multiple orthologs.

Computational methods of identifying novel CRISPR-Cas loci are describedin EP3009511 or US2016208243 and may comprise the following steps:detecting all contigs encoding the Cas1 protein; identifying allpredicted protein coding genes within 20kB of the cas1 gene; comparingthe identified genes with Cas protein-specific profiles and predictingCRISPR arrays; selecting unclassified candidate CRISPR-Cas locicontaining proteins larger than 500 amino acids (>500 aa); analyzingselected candidates using methods such as PSI-BLAST and HHPred to screenfor known protein domains, thereby identifying novel Class 2 CRISPR-Casloci (see also Schmakov et al. 2015, Mol Cell. 60(3):385-97). Inaddition to the above mentioned steps, additional analysis of thecandidates may be conducted by searching metagenomics databases foradditional homologs. Additionally or alternatively, to expand the searchto non-autonomous CRISPR-Cas systems, the same procedure can beperformed with the CRISPR array used as the seed.

In one aspect the detecting all contigs encoding the Cas1 protein isperformed by GenemarkS which a gene prediction program as furtherdescribed in “GeneMarkS: a self-training method for prediction of genestarts in microbial genomes. Implications for finding sequence motifs inregulatory regions.” John Besemer, Alexandre Lomsadze and MarkBorodovsky, Nucleic Acids Research (2001) 29, pp 2607-2618, hereinincorporated by reference.

In one aspect the identifying all predicted protein coding genes iscarried out by comparing the identified genes with Cas protein-specificprofiles and annotating them according to NCBI Conserved Domain Database(CDD) which is a protein annotation resource that consists of acollection of well-annotated multiple sequence alignment models forancient domains and full-length proteins. These are available asposition-specific score matrices (PSSMs) for fast identification ofconserved domains in protein sequences via RPS-BLAST. CDD contentincludes NCBI-curated domains, which use 3D-structure information toexplicitly define domain boundaries and provide insights intosequence/structure/function relationships, as well as domain modelsimported from a number of external source databases (Pfam, SMART, COG,PRK, TIGRFAM). In a further aspect, CRISPR arrays were predicted using aPILER-CR program which is a public domain software for finding CRISPRrepeats as described in “PILER-CR: fast and accurate identification ofCRISPR repeats”, Edgar, R. C., BMC Bioinformatics, January 20;8:18(2007), herein incorporated by reference.

In a further aspect, the case by case analysis is performed usingPSI-BLAST (Position-Specific Iterative Basic Local Alignment SearchTool). PSI-BLAST derives a position-specific scoring matrix (PS SM) orprofile from the multiple sequence alignment of sequences detected abovea given score threshold using protein-protein BLAST. This PSSM is usedto further search the database for new matches, and is updated forsubsequent iterations with these newly detected sequences. Thus,PSI-BLAST provides a means of detecting distant relationships betweenproteins.

In another aspect, the case by case analysis is performed using HHpred,a method for sequence database searching and structure prediction thatis as easy to use as BLAST or PSI-BLAST and that is at the same timemuch more sensitive in finding remote homologs. In fact, HHpred'ssensitivity is competitive with the most powerful servers for structureprediction currently available. HHpred is the first server that is basedon the pairwise comparison of profile hidden Markov models (HMMs).Whereas most conventional sequence search methods search sequencedatabases such as UniProt or the NR, HHpred searches alignmentdatabases, like Pfam or SMART. This greatly simplifies the list of hitsto a number of sequence families instead of a clutter of singlesequences. All major publicly available profile and alignment databasesare available through HHpred. HHpred accepts a single query sequence ora multiple alignment as input. Within only a few minutes it returns thesearch results in an easy-to-read format similar to that of PSI-BLAST.Search options include local or global alignment and scoring secondarystructure similarity. HHpred can produce pairwise query-templatesequence alignments, merged query-template multiple alignments (e.g. fortransitive searches), as well as 3D structural models calculated by theMODELLER software from HHpred alignments.

Orthologs of Cas13

The terms “orthologue” (also referred to as “ortholog” herein) and“homologue” (also referred to as “homolog” herein) are well known in theart. By means of further guidance, a “homologue” of a protein as usedherein is a protein of the same species which performs the same or asimilar function as the protein it is a homologue of. Homologousproteins may but need not be structurally related, or are only partiallystructurally related. An “orthologue” of a protein as used herein is aprotein of a different species which performs the same or a similarfunction as the protein it is an orthologue of. Orthologous proteins maybut need not be structurally related, or are only partially structurallyrelated. Homologs and orthologs may be identified by homology modelling(see, e.g., Greer, Science vol. 228 (1985) 1055, and Blundell et al. EurJ Biochem vol 172 (1988), 513) or “structural BLAST” (Dey F, Cliff ZhangQ, Petrey D, Honig B. Toward a “structural BLAST”: using structuralrelationships to infer function. Protein Sci. 2013 April; 22(4):359-66.doi: 10.1002/pro.2225.). See also Shmakov et al. (2015) for applicationin the field of CRISPR-Cas loci. Homologous proteins may but need not bestructurally related, or are only partially structurally related.

The Cas13 gene is found in several diverse bacterial genomes, typicallyin the same locus with cas1, cas2, and cas4 genes and a CRISPR cassette(for example, FNFX1_1431-FNFX1_1428 of Francisella cf . novicida Fx1).Thus, the layout of this putative novel CRISPR-Cas system appears to besimilar to that of type II-B. Furthermore, similar to Cas9, the Cas13protein contains a readily identifiable C-terminal region that ishomologous to the transposon ORF-B and includes an active RuvC-likenuclease, an arginine-rich region, and a Zn finger (absent in Cas9).However, unlike Cas9, Cas13 is also present in several genomes without aCRISPR-Cas context and its relatively high similarity with ORF-Bsuggests that it might be a transposon component. It was suggested thatif this was a genuine CRISPR-Cas system and Cas13 is a functional analogof Cas9 it would be a novel CRISPR-Cas type, namely type V (SeeAnnotation and Classification of CRISPR-Cas Systems. Makarova K S,Koonin E V. Methods Mol Biol. 2015; 1311:47-75). However, as describedherein, Cas13 is denoted to be in subtype V-A to distinguish it fromC2c1p which does not have an identical domain structure and is hencedenoted to be in subtype V-B.

The present invention encompasses the use of a Cas13 effector protein,derived from a Cas13 locus denoted as subtype V-A. Herein such effectorproteins are also referred to as “Cas13p”, e.g., a Cas13 protein (andsuch effector protein or Cas13 protein or protein derived from a Cas13locus is also called “CRISPR-Cas protein”).

In particular embodiments, the effector protein is a Cas13 effectorprotein from an organism from a genus comprising Streptococcus,Campylobacter, Nitratifractor, Staphylococcus, Parvibaculum, Roseburia,Neisseria, Gluconacetobacter, Azospirillum, Sphaerochaeta,Lactobacillus, Eubacterium, Corynebacter, Carnobacterium, Rhodobacter,Listeria, Paludibacter, Clostridium, Lachnospiraceae, Clostridiaridium,Leptotrichia, Francisella, Legionella, Alicyclobacillus,Methanomethyophilus, Porphyromonas, Prevotella, Bacteroidetes,Helcococcus, Leptospira, Desulfovibrio, Desulfonatronum, Opitutaceae,Tuberibacillus, Bacillus, Brevibacilus, Methylobacterium, Butyvibrio,Perigrinibacterium, Pareubacterium, Moraxella, Thiomicrospira orAcidaminococcus. In particular embodiments, the Cas13 effector proteinis selected from an organism from a genus selected from Eubacterium,Lachnospiraceae, Leptotrichia, Francisella, Methanomethyophilus,Porphyromonas, Prevotella, Leptospira, Butyvibrio, Perigrinibacterium,Pareubacterium, Moraxella, Thiomicrospira or Acidaminococcus

In further particular embodiments, the Cas13 effector protein is from anorganism selected from S. mutans, S. agalactiae, S. equisimilis, S.sanguinis, S. pneumonia; C. jejuni, C. coli; N. salsuginis, N.tergarcus; S. auricularis, S. carnosus; N. meningitides, N. gonorrhoeae;L. monocytogenes, L. ivanovii; C. botulinum, C. difficile, C. tetani, C.sordellii, L. inadai, F. tularensis 1, P. albensis, L. bacterium, B.proteoclasticus, P. bacterium, P. crevioricanis, P. disiens and P.macacae .

The effector protein may comprise a chimeric effector protein comprisinga first fragment from a first effector protein (e.g., a Cas13) orthologand a second fragment from a second effector (e.g., a Cas13) proteinortholog, and wherein the first and second effector protein orthologsare different. At least one of the first and second effector protein(e.g., a Cas13) orthologs may comprise an effector protein (e.g., aCas13) from an organism comprising Streptococcus, Campylobacter,Nitratifractor, Staphylococcus, Parvibaculum, Roseburia, Neisseria,Gluconacetobacter, Azospirillum, Sphaerochaeta, Lactobacillus,Eubacterium, Corynebacter, Carnobacterium, Rhodobacter, Listeria,Paludibacter, Clostridium, Lachnospiraceae, Clostridiaridium,Leptotrichia, Francisella, Legionella, Alicyclobacillus,Methanomethyophilus, Porphyromonas, Prevotella, Bacteroidetes,Helcococcus, Letospira, Desulfovibrio, Desulfonatronum, Opitutaceae,Tuberibacillus, Bacillus, Brevibacilus, Methylobacterium, Butyvibrio,Perigrinibacterium, Pareubacterium, Moraxella, Thiomicrospira orAcidaminococcus; e.g., a chimeric effector protein comprising a firstfragment and a second fragment wherein each of the first and secondfragments is selected from a Cas13 of an organism comprisingStreptococcus, Campylobacter, Nitratifractor, Staphylococcus,Parvibaculum, Roseburia, Neisseria, Gluconacetobacter, Azospirillum,Sphaerochaeta, Lactobacillus, Eubacterium, Corynebacter, Carnobacterium,Rhodobacter, Listeria, Paludibacter, Clostridium, Lachnospiraceae,Clostridiaridium, Leptotrichia, Francisella, Legionella,Alicyclobacillus, Methanomethyophilus, Porphyromonas, Prevotella,Bacteroidetes, Helcococcus, Letospira, Desulfovibrio, Desulfonatronum,Opitutaceae, Tuberibacillus, Bacillus, Brevibacilus, Methylobacterium,Butyvibrio, Perigrinibacterium, Pareubacterium, Moraxella,Thiomicrospira or Acidaminococcus wherein the first and second fragmentsare not from the same bacteria; for instance a chimeric effector proteincomprising a first fragment and a second fragment wherein each of thefirst and second fragments is selected from a Cas13 of S. mutans, S.agalactiae, S. equisimilis, S. sanguinis, S. pneumonia; C. jejuni, C.coli; N. salsuginis, N. tergarcus; S. auricularis, S. carnosus; N.meningitides, N. gonorrhoeae; L. monocytogenes, L. ivanovii; C.botulinum, C. difficile, C. tetani, C. sordellii; Francisella tularensis1, Prevotella albensis, Lachnospiraceae bacterium MC2017 1, Butyrivibrioproteoclasticus, Peregrinibacteria bacterium GW2011_GWA2_33_10,Parcubacteria bacterium GW2011_GWC2_44_17, Smithella sp. SCADC,Acidaminococcus sp. BV3L6, Lachnospiraceae bacterium MA2020, CandidatusMethanoplasma termitum, Eubacterium eligens, Moraxella bovoculi 237,Leptospira inadai, Lachnospiraceae bacterium ND2006, Porphyromonascrevioricanis 3, Prevotella disiens and Porphyromonas macacae, whereinthe first and second fragments are not from the same bacteria.

In a more preferred embodiment, the Cas13p is derived from a bacterialspecies selected from Francisella tularensis 1, Prevotella albensis,Lachnospiraceae bacterium MC2017 1, Butyrivibrio proteoclasticus,Peregrinibacteria bacterium GW2011_GWA2_33_10, Parcubacteria bacteriumGW2011_GWC2_44_17, Smithella sp. SCADC, Acidaminococcus sp. BV3L6,Lachnospiraceae bacterium MA2020, Candidatus Methanoplasma termitum,Eubacterium eligens, Moraxella bovoculi 237, Moraxella bovoculiAAX08_00205, Moraxella bovoculi AAX11 00205, Butyrivibrio sp. NC3005,Thiomicrospira sp. XS5, Leptospira inadai, Lachnospiraceae bacteriumND2006, Porphyromonas crevioricanis 3, Prevotella disiens andPorphyromonas macacae. In certain embodiments, the Cas13p is derivedfrom a bacterial species selected from Acidaminococcus sp. BV3L6,Lachnospiraceae bacterium MA2020. In certain embodiments, the effectorprotein is derived from a subspecies of Francisella tularensis 1,including but not limited to Francisella tularensis subsp. Novicida. Incertain preferred embodiments, the Cas13p is derived from a bacterialspecies selected from Acidaminococcus sp. BV3L6, Lachnospiraceaebacterium ND2006, Lachnospiraceae bacterium MA2020, Moraxella bovoculiAAX08 00205, Moraxella bovoculi AAX11 00205, Butyrivibrio sp. NC3005, orThiomicrospira sp. XS5.

In particular embodiments, the homologue or orthologue of Cas13 asreferred to herein has a sequence homology or identity of at least 80%,more preferably at least 85%, even more preferably at least 90%, such asfor instance at least 95% with the example Cas13 proteins disclosedherein. In further embodiments, the homologue or orthologue of Cas13 asreferred to herein has a sequence identity of at least 80%, morepreferably at least 85%, even more preferably at least 90%, such as forinstance at least 95% with the wild type Cas13. Where the Cas13 has oneor more mutations (mutated), the homologue or orthologue of said Cas13as referred to herein has a sequence identity of at least 80%, morepreferably at least 85%, even more preferably at least 90%, such as forinstance at least 95% with the mutated Cas13.

In an embodiment, the Cas13 protein may be an ortholog of an organism ofa genus which includes, but is not limited to Acidaminococcus sp,Lachnospiraceae bacterium or Moraxella bovoculi; in particularembodiments, the type V Cas protein may be an ortholog of an organism ofa species which includes, but is not limited to Acidaminococcus sp.BV3L6; Lachnospiraceae bacterium ND2006 (LbCas13) or Moraxella bovoculi237. In particular embodiments, the homologue or orthologue of Cas13 asreferred to herein has a sequence homology or identity of at least 80%,more preferably at least 85%, even more preferably at least 90%, such asfor instance at least 95% with one or more of the Cas13 sequencesdisclosed herein. In further embodiments, the homologue or orthologue ofCas13 as referred to herein has a sequence identity of at least 80%,more preferably at least 85%, even more preferably at least 90%, such asfor instance at least 95% with the wild type FnCas13, AsCas13 orLbCas13.

In particular embodiments, the Cas13 protein of the invention has asequence homology or identity of at least 60%, more particularly atleast 70, such as at least 80%, more preferably at least 85%, even morepreferably at least 90%, such as for instance at least 95% with FnCas13,AsCas13 or LbCas13. In further embodiments, the Cas13 protein asreferred to herein has a sequence identity of at least 60%, such as atleast 70%, more particularly at least 80%, more preferably at least 85%,even more preferably at least 90%, such as for instance at least 95%with the wild type AsCas13 or LbCas13. In particular embodiments, theCas13 protein of the present invention has less than 60% sequenceidentity with FnCas13. The skilled person will understand that thisincludes truncated forms of the Cas13 protein whereby the sequenceidentity is determined over the length of the truncated form. Inparticular embodiments, the Cas13 enzyme is not FnCas13.

Modified Cas13 Enzymes

In particular embodiments, it is of interest to make use of anengineered Cas13 protein as defined herein, such as Cas13, wherein theprotein complexes with a nucleic acid molecule comprising RNA to form aCRISPR complex, wherein when in the CRISPR complex, the nucleic acidmolecule targets one or more target polynucleotide loci, the proteincomprises at least one modification compared to unmodified Cas13protein, and wherein the CRISPR complex comprising the modified proteinhas altered activity as compared to the complex comprising theunmodified Cas13 protein. It is to be understood that when referringherein to CRISPR “protein”, the Cas13 protein preferably is a modifiedCRISPR-Cas protein (e.g. having increased or decreased (or no) enzymaticactivity, such as without limitation including Cas13. The term “CRISPRprotein” may be used interchangeably with “CRISPR-Cas protein”,irrespective of whether the CRISPR protein has altered, such asincreased or decreased (or no) enzymatic activity, compared to the wildtype CRISPR protein.

Computational analysis of the primary structure of Cas13 nucleasesreveals three distinct regions. First a C-terminal RuvC like domain,which is the only functional characterized domain. Second a N-terminalalpha-helical region and thirst a mixed alpha and beta region, locatedbetween the RuvC like domain and the alpha-helical region.

Several small stretches of unstructured regions are predicted within theCas13 primary structure. Unstructured regions, which are exposed to thesolvent and not conserved within different Cas13 orthologs, arepreferred sides for splits and insertions of small protein sequences .In addition, these sides can be used to generate chimeric proteinsbetween Cas13 orthologs.

Based on the above information, mutants can be generated which lead toinactivation of the enzyme or which modify the double strand nuclease tonickase activity. In alternative embodiments, this information is usedto develop enzymes with reduced off-target effects (described elsewhereherein)

In certain of the above-described Cas13 enzymes, the enzyme is modifiedby mutation of one or more residues (in the RuvC domain) including butnot limited to positions R909, R912, R930, R947, K949, R951, R955, K965,K968, K1000, K1002, R1003, K1009, K1017, K1022, K1029, K1035, K1054,K1072, K1086, R1094, K1095, K1109, K1118, K1142, K1150, K1158, K1159,R1220, R1226, R1242, and/or R1252 with reference to amino acid positionnumbering of AsCas13 (Acidaminococcus sp. BV3L6). In certainembodiments, the Cas13 enzymes comprising said one or more mutationshave modified, more preferably increased specificity for the target.

In certain of the above-described non-naturally-occurring CRISPR-Casproteins, the enzyme is modified by mutation of one or more residues (inthe RAD50) domain including but not limited positions K324, K335, K337,R331, K369, K370, R386, R392, R393, K400, K404, K406, K408, K414, K429,K436, K438, K459, K460, K464, R670, K675, R681, K686, K689, R699, K705,R725, K729, K739, K748, and/or K752 with reference to amino acidposition numbering of AsCas13 (Acidaminococcus sp. BV3L6). In certainembodiments, the Cas13 enzymes comprising said one or more mutationshave modified, more preferably increased specificity for the target.

In certain of the Cas13 enzymes, the enzyme is modified by mutation ofone or more residues including but not limited positions R912, T923,R947, K949, R951, R955, K965, K968, K1000, R1003, K1009, K1017, K1022,K1029, K1072, K1086, F1103, R1226, and/or R1252 with reference to aminoacid position numbering of AsCas13 (Acidaminococcus sp. BV3L6). Incertain embodiments, the Cas13 enzymes comprising said one or moremutations have modified, more preferably increased specificity for thetarget.

In certain embodiments, the Cas13 enzyme is modified by mutation of oneor more residues including but not limited positions R833, R836, K847,K879, K881, R883, R887, K897, K900, K932, R935, K940, K948, K953, K960,K984, K1003, K1017, R1033, R1138, R1165, and/or R1252 with reference toamino acid position numbering of LbCas13 (Lachnospiraceae bacteriumND2006). In certain embodiments, the Cas13 enzymes comprising said oneor more mutations have modified, more preferably increased specificityfor the target.

In certain embodiments, the Cas13 enzyme is modified by mutation of oneor more residues including but not limited positions K15, R18, K26, Q34,R43, K48, K51, R56, R84, K85, K87, N93, R103, N104, T118, K123, K134,R176, K177, R192, K200, K226, K273, K275, T291, R301, K307, K369, 5404,V409, K414, K436, K438, K468, D482, K516, R518, K524, K530, K532, K548,K559, K570, R574, K592, D596, K603, K607, K613, C647, R681, K686, H720,K739, K748, K757, T766, K780, R790, P791, K796, K809, K815, T816, K860,R862, R863, K868, K897, R909, R912, T923, R947, K949, R951, R955, K965,K968, K1000, R1003, K1009, K1017, K1022, K1029, A1053, K1072, K1086,F1103, S1209, R1226, R1252, K1273, K1282, and/or K1288 with reference toamino acid position numbering of AsCas13 (Acidaminococcus sp. BV3L6). Incertain embodiments, the Cas13 enzymes comprising said one or moremutations have modified, more preferably increased specificity for thetarget.

In certain embodiments, the enzyme is modified by mutation of one ormore residues including but not limited positions K15, R18, K26, R34,R43, K48, K51, K56, K87, K88, D90, K96, K106, K107, K120, Q125, K143,R186, K187, R202, K210, K235, K296, K298, K314, K320, K326, K397, K444,K449, E454, A483, E491, K527, K541, K581, R583, K589, K595, K597, K613,K624, K635, K639, K656, K660, K667, K671, K677, K719, K725, K730, K763,K782, K791, R800, K809, K823, R833, K834, K839, K852, K858, K859, K869,K871, R872, K877, K905, R918, R921, K932, 1960, K962, R964, R968, K978,K981, K1013, R1016, K1021, K1029, K1034, K1041, K1065, K1084, and/orK1098 with reference to amino acid position numbering of FnCas13(Francisella novicida U112). In certain embodiments, the Cas13 enzymescomprising said one or more mutations have modified, more preferablyincreased specificity for the target.

In certain embodiments, the enzyme is modified by mutation of one ormore residues including but not limited positions K15, R18, K26, K34,R43, K48, K51, R56, K83, K84, R86, K92, R102, K103, K116, K121, R158,E159, R174, R182, K206, K251, K253, K269, K271, K278, P342, K380, R385,K390, K415, K421, K457, K471, A506, R508, K514, K520, K522, K538, Y548,K560, K564, K580, K584, K591, K595, K601, K634, K640, R645, K679, K689,K707, T716, K725, R737, R747, R748, K753, K768, K774, K775, K785, K787,R788, Q793, K821, R833, R836, K847, K879, K881, R883, R887, K897, K900,K932, R935, K940, K948, K953, K960, K984, K1003, K1017, R1033, K1121,R1138, R1165, K1190, K1199, and/or K1208 with reference to amino acidposition numbering of LbCas13 (Lachnospiraceae bacterium ND2006). Incertain embodiments, the Cas13 enzymes comprising said one or moremutations have modified, more preferably increased specificity for thetarget.

In certain embodiments, the enzyme is modified by mutation of one ormore residues including but not limited positions K14, R17, R25, K33,M42, Q47, K50, D55, K85, N86, K88, K94, R104, K105, K118, K123, K131,R174, K175, R190, R198,1221, K267, Q269, K285, K291, K297, K357, K403,K409, K414, K448, K460, K501, K515, K550, R552, K558, K564, K566, K582,K593, K604, K608, K623, K627, K633, K637, E643, K780, Y787, K792, K830,Q846, K858, K867, K876, K890, R900, K901, M906, K921, K927, K928, K937,K939, R940, K945, Q975, R987, R990, K1001, R1034, 11036, R1038, R1042,K1052, K1055, K1087, R1090, K1095, N1103, K1108, K1115, K1139, K1158,R1172, K1188, K1276, R1293, A1319, K1340, K1349, and/or K1356 withreference to amino acid position numbering of MbCas13 (Moraxellabovoculi 237). In certain embodiments, the Cas13 enzymes comprising saidone or more mutations have modified, more preferably increasedspecificity for the target.

In one embodiment, the Cas13 protein is modified with a mutation at51228 (e.g., 51228A) with reference to amino acid position numbering ofAsCas13. See Yamano et al., Cell 165:949-962 (2016), which isincorporated herein by reference in its entirety.

In certain embodiments, the Cas13 protein has been modified to recognizea non-natural PAM, such as recognizing a PAM having a sequence orcomprising a sequence YCN, YCV, AYV, TYV, RYN, RCN, TGYV, NTTN, TTN,TRTN, TYTV, TYCT, TYCN, TRTN, NTTN, TACT, TYCC, TRTC, TATV, NTTV, TTV,TSTG, TVTS, TYYS, TCYS, TBYS, TCYS, TNYS, TYYS, TNTN, TSTG, TTCC, TCCC,TATC, TGTG, TCTG, TYCV, or TCTC. In particular embodiments, said mutatedCas13 comprises one or more mutated amino acid residue at position 11,12, 13, 14, 15, 16, 17, 34, 36, 39, 40, 43, 46, 47, 50, 54, 57, 58, 111,126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 157, 158, 159,160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173,174, 175, 176, 177, 178, 532, 533, 534, 535, 536, 537, 538, 539, 540,541, 542, 543, 544, 545, 546, 547, 548, 549, 550, 551, 552, 553, 554,555, 556, 565, 566, 567, 568, 569, 570, 571, 572, 573, 574, 575, 592,593, 594, 595, 596, 597, 598, 599, 600, 601, 602, 603, 604, 605, 606,607, 608, 609, 610, 611, 612, 613, 614, 615, 616, 617, 618, 619, 620,626, 627, 628, 629, 630, 631, 632, 633, 634, 635, 636, 637, 638, 642,643, 644, 645, 646, 647, 648, 649, 651, 652, 653, 654, 655, 656, 676,679, 680, 682, 683, 684, 685, 686, 687, 688, 689, 690, 691, 692, 693,707, 711, 714, 715, 716, 717, 718, 719, 720, 721, 722, 739, 765, 768,769, 773, 777, 778, 779, 780, 781, 782, 783, 784, 785, 786, 871, 872,873, 874, 875, 876, 877, 878, 879, 880, 881, 882, 883, 884, or 1048 ofAsCas13 or a position corresponding thereto in a Cas13 ortholog;preferably, one or more mutated amino acid residue at position 130, 131,132, 133, 134, 135, 136, 162, 163, 164, 165, 166, 167, 168, 169, 170,171, 172, 173, 174, 175, 176, 177, 536, 537, 538, 539, 540, 541, 542,543, 544, 545, 546, 547, 548, 549, 550, 551, 552, 570, 571, 572, 573,595, 596, 597, 598, 599, 600, 601, 602, 603, 604, 605, 606, 607, 608,609, 610, 611, 612, 613, 614, 615, 630, 631, 632, 646, 647, 648, 649,650, 651, 652, 653, 683, 684, 685, 686, 687, 688, 689, or 690;

In certain embodiments, the Cas13 protein is modified to have increasedactivity, i.e. wider PAM specificity. In particular embodiments, theCas13 protein is modified by mutation of one or more residues includingbut not limited positions 539, 542, 547, 548, 550, 551, 552, 167, 604,and/or 607 of AsCas13, or the corresponding position of an AsCas13orthologue, homologue, or variant, preferably mutated amino acidresidues at positions 542 or 542 and 607, wherein said mutationspreferably are 542R and 607R, such as S542R and K607R; or preferablymutated amino acid residues at positions 542 and 548 (and optionally552), wherein said mutations preferably are 542R and 548V (andoptionally 552R), such as S542R and K548V (and optionally N552R); or atposition 532, 538, 542, and/or 595 of LbCas13, or the correspondingposition of an AsCas13 orthologue, homologue, or variant, preferablymutated amino acid residues at positions 532 or 532 and 595, whereinsaid mutations preferably are 532R and 595R, such as G532R and K595R; orpreferably mutated amino acid residues at positions 532 and 538 (andoptionally 542), wherein said mutations preferably are 532R and 538V(and optionally 542R), such as G532R and K538V (and optionally Y542R),most preferably wherein said mutations are S542R and K607R, S542R andK548V, or S542R, K548V and N552R of AsCas13.

Deactivated/Inactivated Cas13 Protein

Where the Cas13 protein has nuclease activity, the Cas13 protein may bemodified to have diminished nuclease activity e.g., nucleaseinactivation of at least 70%, at least 80%, at least 90%, at least 95%,at least 97%, or 100% as compared with the wild type enzyme; or to putin another way, a Cas13 enzyme having advantageously about 0% of thenuclease activity of the non-mutated or wild type Cas13 enzyme orCRISPR-Cas protein, or no more than about 3% or about 5% or about 10% ofthe nuclease activity of the non-mutated or wild type Cas13 enzyme, e.g.of the non-mutated or wild type Francisella novicida U112 (FnCas13),Acidaminococcus sp. BV3L6 (AsCas13), Lachnospiraceae bacterium ND2006(LbCas13) or Moraxella bovoculi 237 (MbCas13 Cas13 enzyme or CRISPR-Casprotein. This is possible by introducing mutations into the nucleasedomains of the Cas13 and orthologs thereof.

In preferred embodiments of the present invention at least one Cas13protein is used which is a Cas13 nickase. More particularly, a Cas13nickase is used which does not cleave the target strand but is capableof cleaving only the strand which is complementary to the target strand,i.e. the non-target DNA strand also referred to herein as the strandwhich is not complementary to the guide sequence. More particularly theCas13 nickase is a Cas13 protein which comprises a mutation in thearginine at position 1226A in the Nuc domain of Cas13 fromAcidaminococcus sp., or a corresponding position in a Cas13 ortholog. Infurther particular embodiments, the enzyme comprises anarginine-to-alanine substitution or an R1226A mutation. It will beunderstood by the skilled person that where the enzyme is not AsCas13, amutation may be made at a residue in a corresponding position. Inparticular embodiments, the Cas13 is FnCas13 and the mutation is at thearginine at position R1218. In particular embodiments, the Cas13 isLbCas13 and the mutation is at the arginine at position R1138. Inparticular embodiments, the Cas13 is MbCas13 and the mutation is at thearginine at position R1293.

In certain embodiments, use is made additionally or alternatively of aCRISPR-Cas protein which is engineered and can comprise one or moremutations that reduce or eliminate a nuclease activity. The amino acidpositions in the FnCas13p RuvC domain include but are not limited toD917A, E1006A, E1028A, D1227A, D1255A, N1257A, D917A, E1006A, E1028A,D1227A, D1255A and N1257A. Applicants have also identified a putativesecond nuclease domain which is most similar to PD−(D/E)XK nucleasesuperfamily and HincII endonuclease like. The point mutations to begenerated in this putative nuclease domain to substantially reducenuclease activity include but are not limited to N580A, N584A, T587A,W609A, D610A, K613A, E614A, D616A, K624A, D625A, K627A and Y629A. In apreferred embodiment, the mutation in the FnCas13p RuvC domain is D917Aor E1006A, wherein the D917A or E1006A mutation completely inactivatesthe DNA cleavage activity of the FnCas13 effector protein. In anotherembodiment, the mutation in the FnCas13p RuvC domain is D1255A, whereinthe mutated FnCas13 effector protein has significantly reducednucleolytic activity.

More particularly, the inactivated Cas13 enzymes include enzymes mutatedin amino acid positions As908, As993, As1263 of AsCas13 or correspondingpositions in Cas13 orthologs. Additionally, the inactivated Cas13enzymes include enzymes mutated in amino acid position Lb832, 925, 947or 1180 of LbCas13 or corresponding positions in Cas13 orthologs. Moreparticularly, the inactivated Cas13 enzymes include enzymes comprisingone or more of mutations AsD908A, AsE993A, AsD1263A of AsCas13 orcorresponding mutations in Cas13 orthologs. Additionally, theinactivated Cas13 enzymes include enzymes comprising one or more ofmutations LbD832A, E925A, D947A or D1180A of LbCas13 or correspondingmutations in Cas13 orthologs.

Mutations can also be made at neighboring residues, e.g., at amino acidsnear those indicated above that participate in the nuclease activity. Insome embodiments, only the RuvC domain is inactivated, and in otherembodiments, another putative nuclease domain is inactivated, whereinthe effector protein complex functions as a nickase and cleaves only oneDNA strand. In a preferred embodiment, the other putative nucleasedomain is a HincII-like endonuclease domain.

The inactivated Cas13 or Cas13 nickase may have associated (e.g., viafusion protein) one or more functional domains, including for example,an adenosine deaminase or catalytic domain thereof. In some cases it isadvantageous that additionally at least one heterologous NLS isprovided. In some instances, it is advantageous to position the NLS atthe N terminus. In general, the positioning of the one or morefunctional domain on the inactivated Cas13 or Cas13 nickase is one whichallows for correct spatial orientation for the functional domain toaffect the target with the attributed functional effect. For example,when the functional domain is an adenosine deaminase catalytic domainthereof, the adenosine deaminase catalytic domain is placed in a spatialorientation which allows it to contact and deaminate a target adenine.This may include positions other than the N-/C-terminus of Cas13. Insome embodiments, the adenosine deaminase protein or catalytic domainthereof is inserted into an internal loop of Cas13.

In certain embodiments, the effector protein (CRISPR enzyme; Cas13;effector protein) according to the invention as described herein is acatalytically inactive or dead Cas13 effector protein (dCas13). In someembodiments, the dCas13 effector comprises mutations in the nucleasedomain. In some embodiments, the dCas13 effector protein has beentruncated. In some embodiments, the dCas13 comprises a truncated form ofa Cas13 effector protein at one or more of the following positions: theC-terminus of the Cas13 effector protein, the N-terminus of the Cas13effector protein, or one or more domains (e.g., an HEPN domain) of aCas13 effector protein. For example, the dCas13 may comprise a truncatedform of a Cas13 effector protein at the C-terminus of the Cas13 effectorprotein. For example, the dCas13 may comprise a truncated form of aCas13 effector protein at the N-terminus of the Cas13 effector protein.For example, the dCas13 may comprise a truncated form of a Cas13effector protein at HEPN domain of the Cas13 effector protein. In someembodiments, such truncations may reduce the size of a fusion protein ofthe Cas13 effector and the one or more functional components. In somecases, the truncated Cas13 may still maintain its RNA binding function.

In some embodiments, the C-terminus of the Cas13 effector can betruncated. For example, at least 20 amino acids, at least 40 aminoacids, at least 50 amino acids, at least 60 amino acids, at least 80amino acids, at least 100 amino acids, at least 120 amino acids, atleast 140 amino acids, at least 150 amino acids, at least 160 aminoacids, at least 180 amino acids, at least 200 amino acids, at least 220amino acids, at least 240 amino acids, at least 250 amino acids, atleast 260 amino acids, or at least 300 amino acids, or at least 350amino acids, or up to 120 amino acids, or up to 140 amino acids, or upto 160 amino acids, or up to 180 amino acids, or up to 200 amino acids,or up to 250 amino acids, or up to 300 amino acids, or up to 350 aminoacids, or up to 400 amino acids, may be truncated at the C-terminus ofthe Cas13 effector. Examples of Cas13 truncations include C-terminalΔ984-1090, C-terminal Δ1026-1090, and C-terminal Δ1053-1090, C-terminalΔ934-1090, C-terminal Δ884-1090, C-terminal Δ834-1090, C-terminalΔ784-1090, and C-terminal Δ734-1090, wherein amino acid positionscorrespond to amino acid positions of Prevotella sp. P5-125 Cas13bprotein. Examples of Cas13 truncations also include C-terminalΔ795-1095, wherein amino acid positions correspond to amino acidpositions of Riemerella anatipestifer Cas13b protein. Examples of Cas13truncations further include C-terminal Δ875-1175, C-terminal Δ895-1175,C-terminal Δ915-1175, C-terminal Δ935-1175, C-terminal Δ955-1175,C-terminal Δ975-1175, C-terminal Δ995-1175, C-terminal Δ1015-1175,C-terminal Δ1035-1175, C-terminal Δ1055-1175, C-terminal Δ1075-1175,C-terminal Δ1095-1175, C-terminal Δ1115-1175, C-terminal Δ1135-1175,C-terminal Δ1155-1175, wherein amino acid positions correspond to aminoacid positions of Porphyromonas gulae Cas13b protein. The skilled personwill understand that similar truncations can be designed for otherCas13b orthologues, or other Cas13 types or subtypes, such as Cas13a,Cas13c, or Cas13d.

In some embodiments, the N-terminus of the Cas13 effector protein may betruncated. For example, at least 20 amino acids, at least 40 aminoacids, at least 50 amino acids, at least 60 amino acids, at least 80amino acids, at least 100 amino acids, at least 120 amino acids, atleast 140 amino acids, at least 150 amino acids, at least 160 aminoacids, at least 180 amino acids, at least 200 amino acids, at least 220amino acids, at least 240 amino acids, at least 250 amino acids, atleast 260 amino acids, or at least 300 amino acids, or at least 350amino acids, or up to 120 amino acids, or up to 140 amino acids, or upto 160 amino acids, or up to 180 amino acids, or up to 200 amino acids,or up to 250 amino acids, or up to 300 amino acids, or up to 350 aminoacids, or up to 400 amino acids, may be truncated at the N-terminus ofthe Cas13 effector. Examples of Cas13 truncations include N-terminalΔ1-125, N-terminal Δ1-88, or N-terminal Δ1-72, wherein amino acidpositions of the truncations correspond to amino acid positions ofPrevotella sp. P5-125 Cas13b protein.

In some embodiments, both the N- and the C-termini of the Cas13 effectorprotein may be truncated. For example, at least 20 amino acids may betruncated at the C-terminus of the Cas13 effector, and at least 20 aminoacids, at least 40 amino acids, at least 60 amino acids, at least 80amino acids, at least 100 amino acids, at least 120 amino acids, atleast 140 amino acids, at least 160 amino acids, at least 180 aminoacids, at least 200 amino acids, at least 220 amino acids, at least 240amino acids, at least 260 amino acids, at least 300 amino acids, or atleast 350 amino acids may be truncated at the N-terminus of the Cas13effector. For example, at least 40 amino acids may be truncated at theC-terminus of the Cas13 effector, and at least 20 amino acids, at least40 amino acids, at least 60 amino acids, at least 80 amino acids, atleast 100 amino acids, at least 120 amino acids, at least 140 aminoacids, at least 160 amino acids, at least 180 amino acids, at least 200amino acids, at least 220 amino acids, at least 240 amino acids, atleast 260 amino acids, at least 300 amino acids, or at least 350 aminoacids may be truncated at the N-terminus of the Cas13 effector. Forexample, at least 60 amino acids may be truncated at the C-terminus ofthe Cas13 effector, and at least 20 amino acids, at least 40 aminoacids, at least 60 amino acids, at least 80 amino acids, at least 100amino acids, at least 120 amino acids, at least 140 amino acids, atleast 160 amino acids, at least 180 amino acids, at least 200 aminoacids, at least 220 amino acids, at least 240 amino acids, at least 260amino acids, at least 300 amino acids, or at least 350 amino acids maybe truncated at the N-terminus of the Cas13 effector. For example, atleast 80 amino acids may be truncated at the C-terminus of the Cas13effector, and at least 20 amino acids, at least 40 amino acids, at least60 amino acids, at least 80 amino acids, at least 100 amino acids, atleast 120 amino acids, at least 140 amino acids, at least 160 aminoacids, at least 180 amino acids, at least 200 amino acids, at least 220amino acids, at least 240 amino acids, at least 260 amino acids, atleast 300 amino acids, or at least 350 amino acids may be truncated atthe N-terminus of the Cas13 effector. For example, at least 100 aminoacids may be truncated at the C-terminus of the Cas13 effector, and atleast 20 amino acids, at least 40 amino acids, at least 60 amino acids,at least 80 amino acids, at least 100 amino acids, at least 120 aminoacids, at least 140 amino acids, at least 160 amino acids, at least 180amino acids, at least 200 amino acids, at least 220 amino acids, atleast 240 amino acids, at least 260 amino acids, at least 300 aminoacids, or at least 350 amino acids may be truncated at the N-terminus ofthe Cas13 effector. For example, at least 120 amino acids may betruncated at the C-terminus of the Cas13 effector, and at least 20 aminoacids, at least 40 amino acids, at least 60 amino acids, at least 80amino acids, at least 100 amino acids, at least 120 amino acids, atleast 140 amino acids, at least 160 amino acids, at least 180 aminoacids, at least 200 amino acids, at least 220 amino acids, at least 240amino acids, at least 260 amino acids, at least 300 amino acids, or atleast 350 amino acids may be truncated at the N-terminus of the Cas13effector. For example, at least 140 amino acids may be truncated at theC-terminus of the Cas13 effector, and at least 20 amino acids, at least40 amino acids, at least 60 amino acids, at least 80 amino acids, atleast 100 amino acids, at least 120 amino acids, at least 140 aminoacids, at least 160 amino acids, at least 180 amino acids, at least 200amino acids, at least 220 amino acids, at least 240 amino acids, atleast 260 amino acids, at least 300 amino acids, or at least 350 aminoacids may be truncated at the N-terminus of the Cas13 effector. Forexample, at least 160 amino acids may be truncated at the C-terminus ofthe Cas13 effector, and at least 20 amino acids, at least 40 aminoacids, at least 60 amino acids, at least 80 amino acids, at least 100amino acids, at least 120 amino acids, at least 140 amino acids, atleast 160 amino acids, at least 180 amino acids, at least 200 aminoacids, at least 220 amino acids, at least 240 amino acids, at least 260amino acids, at least 300 amino acids, or at least 350 amino acids maybe truncated at the N-terminus of the Cas13 effector. For example, atleast 180 amino acids may be truncated at the C-terminus of the Cas13effector, and at least 20 amino acids, at least 40 amino acids, at least60 amino acids, at least 80 amino acids, at least 100 amino acids, atleast 120 amino acids, at least 140 amino acids, at least 160 aminoacids, at least 180 amino acids, at least 200 amino acids, at least 220amino acids, at least 240 amino acids, at least 260 amino acids, atleast 300 amino acids, or at least 350 amino acids may be truncated atthe N-terminus of the Cas13 effector. For example, at least 200 aminoacids may be truncated at the C-terminus of the Cas13 effector, and atleast 20 amino acids, at least 40 amino acids, at least 60 amino acids,at least 80 amino acids, at least 100 amino acids, at least 120 aminoacids, at least 140 amino acids, at least 160 amino acids, at least 180amino acids, at least 200 amino acids, at least 220 amino acids, atleast 240 amino acids, at least 260 amino acids, at least 300 aminoacids, or at least 350 amino acids may be truncated at the N-terminus ofthe Cas13 effector. For example, at least 220 amino acids may betruncated at the C-terminus of the Cas13 effector, and at least 20 aminoacids, at least 40 amino acids, at least 60 amino acids, at least 80amino acids, at least 100 amino acids, at least 120 amino acids, atleast 140 amino acids, at least 160 amino acids, at least 180 aminoacids, at least 200 amino acids, at least 220 amino acids, at least 240amino acids, at least 260 amino acids, at least 300 amino acids, or atleast 350 amino acids may be truncated at the N-terminus of the Cas13effector. For example, at least 240 amino acids may be truncated at theC-terminus of the Cas13 effector, and at least 20 amino acids, at least40 amino acids, at least 60 amino acids, at least 80 amino acids, atleast 100 amino acids, at least 120 amino acids, at least 140 aminoacids, at least 160 amino acids, at least 180 amino acids, at least 200amino acids, at least 220 amino acids, at least 240 amino acids, atleast 260 amino acids, at least 300 amino acids, or at least 350 aminoacids may be truncated at the N-terminus of the Cas13 effector. Forexample, at least 260 amino acids may be truncated at the C-terminus ofthe Cas13 effector, and at least 20 amino acids, at least 40 aminoacids, at least 60 amino acids, at least 80 amino acids, at least 100amino acids, at least 120 amino acids, at least 140 amino acids, atleast 160 amino acids, at least 180 amino acids, at least 200 aminoacids, at least 220 amino acids, at least 240 amino acids, at least 260amino acids, at least 300 amino acids, or at least 350 amino acids maybe truncated at the N-terminus of the Cas13 effector. For example, atleast 280 amino acids may be truncated at the C-terminus of the Cas13effector, and at least 20 amino acids, at least 40 amino acids, at least60 amino acids, at least 80 amino acids, at least 100 amino acids, atleast 120 amino acids, at least 140 amino acids, at least 160 aminoacids, at least 180 amino acids, at least 200 amino acids, at least 220amino acids, at least 240 amino acids, at least 260 amino acids, atleast 300 amino acids, or at least 350 amino acids may be truncated atthe N-terminus of the Cas13 effector. For example, at least 300 aminoacids may be truncated at the C-terminus of the Cas13 effector, and atleast 20 amino acids, at least 40 amino acids, at least 60 amino acids,at least 80 amino acids, at least 100 amino acids, at least 120 aminoacids, at least 140 amino acids, at least 160 amino acids, at least 180amino acids, at least 200 amino acids, at least 220 amino acids, atleast 240 amino acids, at least 260 amino acids, at least 300 aminoacids, or at least 350 amino acids may be truncated at the N-terminus ofthe Cas13 effector. For example, at least 350 amino acids may betruncated at the C-terminus of the Cas13 effector, and at least 20 aminoacids, at least 40 amino acids, at least 60 amino acids, at least 80amino acids, at least 100 amino acids, at least 120 amino acids, atleast 140 amino acids, at least 160 amino acids, at least 180 aminoacids, at least 200 amino acids, at least 220 amino acids, at least 240amino acids, at least 260 amino acids, at least 300 amino acids, or atleast 350 amino acids may be truncated at the N-terminus of the Cas13effector. For example, at least 20 amino acids may be truncated at theN-terminus of the Cas13 effector, and at least 20 amino acids, at least40 amino acids, at least 60 amino acids, at least 80 amino acids, atleast 100 amino acids, at least 120 amino acids, at least 140 aminoacids, at least 160 amino acids, at least 180 amino acids, at least 200amino acids, at least 220 amino acids, at least 240 amino acids, atleast 260 amino acids, at least 300 amino acids, or at least 350 aminoacids may be truncated at the C-terminus of the Cas13 effector. Forexample, at least 40 amino acids may be truncated at the N-terminus ofthe Cas13 effector, and at least 20 amino acids, at least 40 aminoacids, at least 60 amino acids, at least 80 amino acids, at least 100amino acids, at least 120 amino acids, at least 140 amino acids, atleast 160 amino acids, at least 180 amino acids, at least 200 aminoacids, at least 220 amino acids, at least 240 amino acids, at least 260amino acids, at least 300 amino acids, or at least 350 amino acids maybe truncated at the C-terminus of the Cas13 effector. For example, atleast 60 amino acids may be truncated at the N-terminus of the Cas13effector, and at least 20 amino acids, at least 40 amino acids, at least60 amino acids, at least 80 amino acids, at least 100 amino acids, atleast 120 amino acids, at least 140 amino acids, at least 160 aminoacids, at least 180 amino acids, at least 200 amino acids, at least 220amino acids, at least 240 amino acids, at least 260 amino acids, atleast 300 amino acids, or at least 350 amino acids may be truncated atthe C-terminus of the Cas13 effector. For example, at least 80 aminoacids may be truncated at the N-terminus of the Cas13 effector, and atleast 20 amino acids, at least 40 amino acids, at least 60 amino acids,at least 80 amino acids, at least 100 amino acids, at least 120 aminoacids, at least 140 amino acids, at least 160 amino acids, at least 180amino acids, at least 200 amino acids, at least 220 amino acids, atleast 240 amino acids, at least 260 amino acids, at least 300 aminoacids, or at least 350 amino acids may be truncated at the C-terminus ofthe Cas13 effector. For example, at least 100 amino acids may betruncated at the N-terminus of the Cas13 effector, and at least 20 aminoacids, at least 40 amino acids, at least 60 amino acids, at least 80amino acids, at least 100 amino acids, at least 120 amino acids, atleast 140 amino acids, at least 160 amino acids, at least 180 aminoacids, at least 200 amino acids, at least 220 amino acids, at least 240amino acids, at least 260 amino acids, at least 300 amino acids, or atleast 350 amino acids may be truncated at the C-terminus of the Cas13effector. For example, at least 120 amino acids may be truncated at theN-terminus of the Cas13 effector, and at least 20 amino acids, at least40 amino acids, at least 60 amino acids, at least 80 amino acids, atleast 100 amino acids, at least 120 amino acids, at least 140 aminoacids, at least 160 amino acids, at least 180 amino acids, at least 200amino acids, at least 220 amino acids, at least 240 amino acids, atleast 260 amino acids, at least 300 amino acids, or at least 350 aminoacids may be truncated at the C-terminus of the Cas13 effector. Forexample, at least 140 amino acids may be truncated at the N-terminus ofthe Cas13 effector, and at least 20 amino acids, at least 40 aminoacids, at least 60 amino acids, at least 80 amino acids, at least 100amino acids, at least 120 amino acids, at least 140 amino acids, atleast 160 amino acids, at least 180 amino acids, at least 200 aminoacids, at least 220 amino acids, at least 240 amino acids, at least 260amino acids, at least 300 amino acids, or at least 350 amino acids maybe truncated at the C-terminus of the Cas13 effector. For example, atleast 160 amino acids may be truncated at the N-terminus of the Cas13effector, and at least 20 amino acids, at least 40 amino acids, at least60 amino acids, at least 80 amino acids, at least 100 amino acids, atleast 120 amino acids, at least 140 amino acids, at least 160 aminoacids, at least 180 amino acids, at least 200 amino acids, at least 220amino acids, at least 240 amino acids, at least 260 amino acids, atleast 300 amino acids, or at least 350 amino acids may be truncated atthe C-terminus of the Cas13 effector. For example, at least 180 aminoacids may be truncated at the N-terminus of the Cas13 effector, and atleast 20 amino acids, at least 40 amino acids, at least 60 amino acids,at least 80 amino acids, at least 100 amino acids, at least 120 aminoacids, at least 140 amino acids, at least 160 amino acids, at least 180amino acids, at least 200 amino acids, at least 220 amino acids, atleast 240 amino acids, at least 260 amino acids, at least 300 aminoacids, or at least 350 amino acids may be truncated at the C-terminus ofthe Cas13 effector. For example, at least 200 amino acids may betruncated at the N-terminus of the Cas13 effector, and at least 20 aminoacids, at least 40 amino acids, at least 60 amino acids, at least 80amino acids, at least 100 amino acids, at least 120 amino acids, atleast 140 amino acids, at least 160 amino acids, at least 180 aminoacids, at least 200 amino acids, at least 220 amino acids, at least 240amino acids, at least 260 amino acids, at least 300 amino acids, or atleast 350 amino acids may be truncated at the C-terminus of the Cas13effector. For example, at least 220 amino acids may be truncated at theN-terminus of the Cas13 effector, and at least 20 amino acids, at least40 amino acids, at least 60 amino acids, at least 80 amino acids, atleast 100 amino acids, at least 120 amino acids, at least 140 aminoacids, at least 160 amino acids, at least 180 amino acids, at least 200amino acids, at least 220 amino acids, at least 240 amino acids, atleast 260 amino acids, at least 300 amino acids, or at least 350 aminoacids may be truncated at the C-terminus of the Cas13 effector. Forexample, at least 240 amino acids may be truncated at the N-terminus ofthe Cas13 effector, and at least 20 amino acids, at least 40 aminoacids, at least 60 amino acids, at least 80 amino acids, at least 100amino acids, at least 120 amino acids, at least 140 amino acids, atleast 160 amino acids, at least 180 amino acids, at least 200 aminoacids, at least 220 amino acids, at least 240 amino acids, at least 260amino acids, at least 300 amino acids, or at least 350 amino acids maybe truncated at the C-terminus of the Cas13 effector. For example, atleast 260 amino acids may be truncated at the N-terminus of the Cas13effector, and at least 20 amino acids, at least 40 amino acids, at least60 amino acids, at least 80 amino acids, at least 100 amino acids, atleast 120 amino acids, at least 140 amino acids, at least 160 aminoacids, at least 180 amino acids, at least 200 amino acids, at least 220amino acids, at least 240 amino acids, at least 260 amino acids, atleast 300 amino acids, or at least 350 amino acids may be truncated atthe C-terminus of the Cas13 effector. For example, at least 280 aminoacids may be truncated at the N-terminus of the Cas13 effector, and atleast 20 amino acids, at least 40 amino acids, at least 60 amino acids,at least 80 amino acids, at least 100 amino acids, at least 120 aminoacids, at least 140 amino acids, at least 160 amino acids, at least 180amino acids, at least 200 amino acids, at least 220 amino acids, atleast 240 amino acids, at least 260 amino acids, at least 300 aminoacids, or at least 350 amino acids may be truncated at the C-terminus ofthe Cas13 effector. For example, at least 300 amino acids may betruncated at the N-terminus of the Cas13 effector, and at least 20 aminoacids, at least 40 amino acids, at least 60 amino acids, at least 80amino acids, at least 100 amino acids, at least 120 amino acids, atleast 140 amino acids, at least 160 amino acids, at least 180 aminoacids, at least 200 amino acids, at least 220 amino acids, at least 240amino acids, at least 260 amino acids, at least 300 amino acids, or atleast 350 amino acids may be truncated at the C-terminus of the Cas13effector. For example, at least 350 amino acids may be truncated at theN-terminus of the Cas13 effector, and at least 20 amino acids, at least40 amino acids, at least 60 amino acids, at least 80 amino acids, atleast 100 amino acids, at least 120 amino acids, at least 140 aminoacids, at least 160 amino acids, at least 180 amino acids, at least 200amino acids, at least 220 amino acids, at least 240 amino acids, atleast 260 amino acids, at least 300 amino acids, or at least 350 aminoacids may be truncated at the C-terminus of the Cas13 effector.

CRISPR-Cas Protein and Guide

In the methods and systems of the present invention use is made of aCRISPR-Cas protein and corresponding guide molecule. More particularly,the CRISPR-Cas protein is a class 2 CRISPR-Cas protein. In certainembodiments, said CRISPR-Cas protein is a Cas13. The CRISPR-Cas systemdoes not require the generation of customized proteins to targetspecific sequences but rather a single Cas protein can be programmed byguide molecule to recognize a specific nucleic acid target, in otherwords the Cas enzyme protein can be recruited to a specific nucleic acidtarget locus of interest using said guide molecule.

Guide Molecule

The guide molecule or guide RNA of a Class 2 type V CRISPR-Cas proteincomprises a tracr-mate sequence (encompassing a “direct repeat” in thecontext of an endogenous CRISPR system) and a guide sequence (alsoreferred to as a “spacer” in the context of an endogenous CRISPRsystem). Indeed, in contrast to the type II CRISPR-Cas proteins, theCas13 protein does not rely on the presence of a tracr sequence. In someembodiments, the CRISPR-Cas system or complex as described herein doesnot comprise and/or does not rely on the presence of a tracr sequence(e.g. if the Cas protein is Cas13). In certain embodiments, the guidemolecule may comprise, consist essentially of, or consist of a directrepeat sequence fused or linked to a guide sequence or spacer sequence.

In general, a CRISPR system is characterized by elements that promotethe formation of a CRISPR complex at the site of a target sequence. Inthe context of formation of a CRISPR complex, “target sequence” refersto a sequence to which a guide sequence is designed to havecomplementarity, where hybridization between a target DNA sequence and aguide sequence promotes the formation of a CRISPR complex.

The terms “guide molecule” and “guide RNA” are used interchangeablyherein to refer to RNA-based molecules that are capable of forming acomplex with a CRISPR-Cas protein and comprises a guide sequence havingsufficient complementarity with a target nucleic acid sequence tohybridize with the target nucleic acid sequence and directsequence-specific binding of the complex to the target nucleic acidsequence. The guide molecule or guide RNA specifically encompassesRNA-based molecules having one or more chemically modifications (e.g.,by chemical linking two ribonucleotides or by replacement of one or moreribonucleotides with one or more deoxyribonucleotides), as describedherein.

As used herein, the term “guide sequence” in the context of a CRISPR-Cassystem, comprises any polynucleotide sequence having sufficientcomplementarity with a target nucleic acid sequence to hybridize withthe target nucleic acid sequence and direct sequence-specific binding ofa nucleic acid-targeting complex to the target nucleic acid sequence. Inthe context of the present invention the target nucleic acid sequence ortarget sequence is the sequence comprising the target adenosine to bedeaminated also referred to herein as the “target adenosine”. In someembodiments, except for the intended dA-C mismatch, the degree ofcomplementarity, when optimally aligned using a suitable alignmentalgorithm, is about or more than about 50%, 60%, 75%, 80%, 85%, 90%,95%, 97.5%, 99%, or more. Optimal alignment may be determined with theuse of any suitable algorithm for aligning sequences, non-limitingexample of which include the Smith-Waterman algorithm, theNeedleman-Wunsch algorithm, algorithms based on the Burrows-WheelerTransform (e.g., the Burrows Wheeler Aligner), ClustalW, Clustal X,BLAT, Novoalign (Novocraft Technologies; available atwww.novocraft.com), ELAND (Illumina, San Diego, Calif.), SOAP (availableat soap.genomics.org.cn), and Maq (available at maq.sourceforge.net).The ability of a guide sequence (within a nucleic acid-targeting guideRNA) to direct sequence-specific binding of a nucleic acid-targetingcomplex to a target nucleic acid sequence may be assessed by anysuitable assay. For example, the components of a nucleic acid-targetingCRISPR system sufficient to form a nucleic acid-targeting complex,including the guide sequence to be tested, may be provided to a hostcell having the corresponding target nucleic acid sequence, such as bytransfection with vectors encoding the components of the nucleicacid-targeting complex, followed by an assessment of preferentialtargeting (e.g., cleavage) within the target nucleic acid sequence, suchas by Surveyor assay as described herein. Similarly, cleavage of atarget nucleic acid sequence (or a sequence in the vicinity thereof) maybe evaluated in a test tube by providing the target nucleic acidsequence, components of a nucleic acid-targeting complex, including theguide sequence to be tested and a control guide sequence different fromthe test guide sequence, and comparing binding or rate of cleavage at orin the vicinity of the target sequence between the test and controlguide sequence reactions. Other assays are possible, and will occur tothose skilled in the art. A guide sequence, and hence a nucleicacid-targeting guide RNA may be selected to target any target nucleicacid sequence.

In some embodiments, the guide molecule comprises a guide sequence thatis designed to have at least one mismatch with the target sequence, suchthat an RNA duplex formed between the guide sequence and the targetsequence comprises a non-pairing C in the guide sequence opposite to thetarget A for deamination on the target sequence. In some embodiments,aside from this A-C mismatch, the degree of complementarity, whenoptimally aligned using a suitable alignment algorithm, is about or morethan about 50%, 60%, 75%, 80%, 85%, 90%, 95%, 97.5%, 99%, or more.

In certain embodiments, the guide sequence or spacer length of the guidemolecules is from 15 to 50 nt. In certain embodiments, the spacer lengthof the guide RNA is at least 15 nucleotides. In certain embodiments, thespacer length is from 15 to 17 nt, e.g., 15, 16, or 17 nt, from 17 to 20nt, e.g., 17, 18, 19, or 20 nt, from 20 to 24 nt, e.g., 20, 21, 22, 23,or 24 nt, from 23 to 25 nt, e.g., 23, 24, or 25 nt, from 24 to 27 nt,e.g., 24, 25, 26, or 27 nt, from 27-30 nt, e.g., 27, 28, 29, or 30 nt,from 30-35 nt, e.g., 30, 31, 32, 33, 34, or 35 nt, or 35 nt or longer.In certain example embodiment, the guide sequence is 15, 16, 17,18, 19,20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37,38, 39 40, 41, 42, 43, 44, 45, 46, 47 48, 49, 50, 51, 52, 53, 54, 55,56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73,74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91,92, 93, 94, 95, 96, 97, 98, 99, or 100 nt.

In some embodiments, the guide sequence is an RNA sequence of between 10to 50 nt in length, but more particularly of about 20-30 ntadvantageously about 20 nt, 23-25 nt or 24 nt. The guide sequence isselected so as to ensure that it hybridizes to the target sequencecomprising the adenosine to be deaminated. This is described more indetail below. Selection can encompass further steps which increaseefficacy and specificity of deamination.

In some embodiments, the guide sequence is about 20 nt to about 30 ntlong and hybridizes to the target DNA strand to form an almost perfectlymatched duplex, except for having a dA-C mismatch at the targetadenosine site. Particularly, in some embodiments, the dA-C mismatch islocated close to the center of the target sequence (and thus the centerof the duplex upon hybridization of the guide sequence to the targetsequence), thereby restricting the adenosine deaminase to a narrowediting window (e.g., about 4 bp wide). In some embodiments, the targetsequence may comprise more than one target adenosine to be deaminated.In further embodiments the target sequence may further comprise one ormore dA-C mismatch 3′ to the target adenosine site. In some embodiments,to avoid off-target editing at an unintended Adenine site in the targetsequence, the guide sequence can be designed to comprise a non-pairingGuanine at a position corresponding to said unintended Adenine tointroduce a dA-G mismatch, which is catalytically unfavorable forcertain adenosine deaminases such as ADAR1 and ADAR2. See Wong et al.,RNA 7:846-858 (2001), which is incorporated herein by reference in itsentirety.

In some embodiments, a Cas13 guide sequence having a canonical length(e.g., about 24 nt for AsCas13) is used to form an RNA duplex with thetarget DNA. In some embodiments, a Cas13 guide molecule longer than thecanonical length (e.g., >24 nt for AsCas13) is used to form an RNAduplex with the target DNA including outside of the Cas13-guideRNA-target DNA complex. This can be of interest where deamination ofmore than one adenine within a given stretch of nucleotides is ofinterest. In alternative embodiments, it is of interest to maintain thelimitation of the canonical guide sequence length. In some embodiments,the guide sequence is designed to introduce a dA-C mismatch outside ofthe canonical length of Cas13 guide, which may decrease steric hindranceby Cas13 and increase the frequency of contact between the adenosinedeaminase and the dA-C mismatch.

In some embodiments, the sequence of the guide molecule (direct repeatand/or spacer) is selected to reduce the degree secondary structurewithin the guide molecule. In some embodiments, about or less than about75%, 50%, 40%, 30%, 25%, 20%, 15%, 10%, 5%, 1%, or fewer of thenucleotides of the nucleic acid-targeting guide RNA participate inself-complementary base pairing when optimally folded. Optimal foldingmay be determined by any suitable polynucleotide folding algorithm. Someprograms are based on calculating the minimal Gibbs free energy. Anexample of one such algorithm is mFold, as described by Zuker andStiegler (Nucleic Acids Res. 9 (1981), 133-148). Another example foldingalgorithm is the online webserver RNAfold, developed at Institute forTheoretical Chemistry at the University of Vienna, using the centroidstructure prediction algorithm (see e.g., A. R. Gruber et al., 2008,Cell 106(1): 23-24; and P A Carr and G M Church, 2009, NatureBiotechnology 27(12): 1151-62).

In some embodiments, it is of interest to reduce the susceptibility ofthe guide molecule to RNA cleavage, such as to cleavage by Cas13.Accordingly, in particular embodiments, the guide molecule is adjustedto avoid cleavage by Cas13 or other RNA-cleaving enzymes.

In certain embodiments, the guide molecule comprises non-naturallyoccurring nucleic acids and/or non-naturally occurring nucleotidesand/or nucleotide analogs, and/or chemically modifications. Preferably,these non-naturally occurring nucleic acids and non-naturally occurringnucleotides are located outside the guide sequence. Non-naturallyoccurring nucleic acids can include, for example, mixtures of naturallyand non-naturally occurring nucleotides. Non-naturally occurringnucleotides and/or nucleotide analogs may be modified at the ribose,phosphate, and/or base moiety. In an embodiment of the invention, aguide nucleic acid comprises ribonucleotides and non-ribonucleotides. Inone such embodiment, a guide comprises one or more ribonucleotides andone or more deoxyribonucleotides. In an embodiment of the invention, theguide comprises one or more non-naturally occurring nucleotide ornucleotide analog such as a nucleotide with phosphorothioate linkage, alocked nucleic acid (LNA) nucleotides comprising a methylene bridgebetween the 2′ and 4′ carbons of the ribose ring, or bridged nucleicacids (BNA). Other examples of modified nucleotides include 2′-O-methylanalogs, 2′-deoxy analogs, or 2′-fluoro analogs. Further examples ofmodified bases include, but are not limited to, 2-aminopurine,5-bromo-uridine, pseudouridine, inosine, 7-methylguanosine. Examples ofguide RNA chemical modifications include, without limitation,incorporation of 2′-O-methyl (M), 2′-O-methyl 3′phosphorothioate (MS),S-constrained ethyl(cEt), or 2′-O-methyl 3′thioPACE (MSP) at one or moreterminal nucleotides. Such chemically modified guides can compriseincreased stability and increased activity as compared to unmodifiedguides, though on-target vs. off-target specificity is not predictable.(See, Hendel, 2015, Nat Biotechnol. 33(9):985-9, doi: 10.1038/nbt.3290,published online 29 Jun. 2015 Ragdarm et al., 0215, PNAS, E7110-E7111;Allerson et al., J. Med. Chem. 2005, 48:901-904; Bramsen et al., Front.Genet., 2012, 3:154; Deng et al., PNAS, 2015, 112:11870-11875; Sharma etal., Med Chem Comm., 2014, 5:1454-1471; Hendel et al., Nat. Biotechnol.(2015) 33(9): 985-989; Li et al., Nature Biomedical Engineering, 2017,1, 0066 DOI:10.1038/s41551-017-0066). In some embodiments, the 5′ and/or3′ end of a guide RNA is modified by a variety of functional moietiesincluding fluorescent dyes, polyethylene glycol, cholesterol, proteins,or detection tags. (See Kelly et al., 2016, J. Biotech. 233:74-83). Incertain embodiments, a guide comprises ribonucleotides in a region thatbinds to a target DNA and one or more deoxyribonucletides and/ornucleotide analogs in a region that binds to Cas13. In an embodiment ofthe invention, deoxyribonucleotides and/or nucleotide analogs areincorporated in engineered guide structures, such as, withoutlimitation, stem-loop regions, and the seed region. For Cas13 guide, incertain embodiments, the modification is not in the 5′-handle of thestem-loop regions. Chemical modification in the 5′-handle of thestem-loop region of a guide may abolish its function (see Li, et al.,Nature Biomedical Engineering, 2017, 1:0066). In certain embodiments, atleast 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19,20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50, or 75nucleotides of a guide is chemically modified. In some embodiments, 3-5nucleotides at either the 3′ or the 5′ end of a guide is chemicallymodified. In some embodiments, only minor modifications are introducedin the seed region, such as 2′-F modifications. In some embodiments,2′-F modification is introduced at the 3′ end of a guide. In certainembodiments, three to five nucleotides at the 5′ and/or the 3′ end ofthe guide are chemically modified with 2′-O-methyl (M), 2′-O-methyl 3′phosphorothioate (MS), S-constrained ethyl(cEt), or 2′-O-methyl 3′thioPACE (MSP). Such modification can enhance genome editing efficiency(see Hendel et al., Nat. Biotechnol. (2015) 33(9): 985-989). In certainembodiments, all of the phosphodiester bonds of a guide are substitutedwith phosphorothioates (PS) for enhancing levels of gene disruption. Incertain embodiments, more than five nucleotides at the 5′ and/or the 3′end of the guide are chemically modified with 2′-O-Me, 2′-F orS-constrained ethyl(cEt). Such chemically modified guide can mediateenhanced levels of gene disruption (see Ragdarm et al., 0215, PNAS,E7110-E7111). In an embodiment of the invention, a guide is modified tocomprise a chemical moiety at its 3′ and/or 5′ end. Such moietiesinclude, but are not limited to amine, azide, alkyne, thio,dibenzocyclooctyne (DBCO), or Rhodamine. In certain embodiment, thechemical moiety is conjugated to the guide by a linker, such as an alkylchain. In certain embodiments, the chemical moiety of the modified guidecan be used to attach the guide to another molecule, such as DNA, RNA,protein, or nanoparticles. Such chemically modified guide can be used toidentify or enrich cells generically edited by a CRISPR system (see Leeet al., eLife, 2017, 6:e25312, DOI:10.7554).

In some embodiments, the guide comprises a modified Cas13 crRNA, havinga 5′-handle and a guide segment further comprising a seed region and a3′-terminus. In some embodiments, the modified guide can be used with aCas13 of any one of Acidaminococcus sp. BV3L6 Cas13 (AsCas13);Francisella tularensis subsp. Novicida U112 Cas13 (FnCas13); L.bacterium MC2017 Cas13 (Lb3Cas13); Butyrivibrio proteoclasticus Cas13(BpCas13); Parcubacteria bacterium GWC2011_GWC2_44_17 Cas13 (PbCas13);Peregrinibacteria bacterium GW2011_GWA_33_10 Cas13 (PeCas13); Leptospirainadai Cas13 (LiCas13); Smithella sp. SC_K08D17 Cas13 (SsCas13); L.bacterium MA2020 Cas13 (Lb2Cas13); Porphyromonas crevioricanis Cas13(PcCas13); Porphyromonas macacae Cas13 (PmCas13); CandidatusMethanoplasma termitum Cas13 (CMtCas13); Eubacterium eligens Cas13(EeCas13); Moraxella bovoculi 237 Cas13 (MbCas13); Prevotella disiensCas13 (PdCas13); or L. bacterium ND2006 Cas13 (LbCas13).

In some embodiments, the modification to the guide is a chemicalmodification, an insertion, a deletion or a split. In some embodiments,the chemical modification includes, but is not limited to, incorporationof 2′-O-methyl (M) analogs, 2′-deoxy analogs, 2-thiouridine analogs,N6-methyladenosine analogs, 2′-fluoro analogs, 2-aminopurine,5-bromo-uridine, pseudouridine (Ψ), N1-methylpseudouridine (melΨ),5-methoxyuridine(5moU), inosine, 7-methylguanosine, 2′-O-methyl3′phosphorothioate (MS), S-constrained ethyl(cEt), phosphorothioate(PS), or 2′-O-methyl 3′thioPACE (MSP). In some embodiments, the guidecomprises one or more of phosphorothioate modifications. In certainembodiments, at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15,16, 17, 18, 19, 20, or 25 nucleotides of the guide are chemicallymodified. In certain embodiments, one or more nucleotides in the seedregion are chemically modified. In certain embodiments, one or morenucleotides in the 3′-terminus are chemically modified. In certainembodiments, none of the nucleotides in the 5′-handle is chemicallymodified. In some embodiments, the chemical modification in the seedregion is a minor modification, such as incorporation of a 2′-fluoroanalog. In a specific embodiment, one nucleotide of the seed region isreplaced with a 2′-fluoro analog. In some embodiments, 5 to 10nucleotides in the 3′-terminus are chemically modified. Such chemicalmodifications at the 3′-terminus of the Cas13 CrRNA may improve Cas13activity (see Li, et al., Nature Biomedical Engineering, 2017, 1:0066).In a specific embodiment, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 nucleotides inthe 3′-terminus are replaced with 2′-fluoro analogues. In a specificembodiment, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 nucleotides in the3′-terminus are replaced with 2′-O-methyl (M) analogs.

In some embodiments, the loop of the 5′-handle of the guide is modified.In some embodiments, the loop of the 5′-handle of the guide is modifiedto have a deletion, an insertion, a split, or chemical modifications. Incertain embodiments, the modified loop comprises 3, 4, or 5 nucleotides.In certain embodiments, the loop comprises the sequence of UCUU, UUUU,UAUU, or UGUU.

In some embodiments, the guide molecule forms a stem loop with aseparate non-covalently linked sequence, which can be DNA or RNA. Inparticular embodiments, the sequences forming the guide are firstsynthesized using the standard phosphoramidite synthetic protocol(Herdewijn, P., ed., Methods in Molecular Biology Col 288,Oligonucleotide Synthesis: Methods and Applications, Humana Press, NewJersey (2012)). In some embodiments, these sequences can befunctionalized to contain an appropriate functional group for ligationusing the standard protocol known in the art (Hermanson, G. T.,Bioconjugate Techniques, Academic Press (2013)). Examples of functionalgroups include, but are not limited to, hydroxyl, amine, carboxylicacid, carboxylic acid halide, carboxylic acid active ester, aldehyde,carbonyl, chlorocarbonyl, imidazolylcarbonyl, hydrozide, semicarbazide,thio semicarbazide, thiol, maleimide, haloalkyl, sufonyl, ally,propargyl, diene, alkyne, and azide. Once this sequence isfunctionalized, a covalent chemical bond or linkage can be formedbetween this sequence and the direct repeat sequence. Examples ofchemical bonds include, but are not limited to, those based oncarbamates, ethers, esters, amides, imines, amidines, aminotrizines,hydrozone, disulfides, thioethers, thioesters, phosphorothioates,phosphorodithioates, sulfonamides, sulfonates, fulfones, sulfoxides,ureas, thioureas, hydrazide, oxime, triazole, photolabile linkages, C—Cbond forming groups such as Diels-Alder cyclo-addition pairs orring-closing metathesis pairs, and Michael reaction pairs.

In some embodiments, these stem-loop forming sequences can be chemicallysynthesized. In some embodiments, the chemical synthesis uses automated,solid-phase oligonucleotide synthesis machines with 2′-acetoxyethylorthoester (2′-ACE) (Scaringe et al., J. Am. Chem. Soc. (1998) 120:11820-11821; Scaringe, Methods Enzymol. (2000) 317: 3-18) or2′-thionocarbamate (2′-TC) chemistry (Dellinger et al., J. Am. Chem.Soc. (2011) 133: 11540-11546; Hendel et al., Nat. Biotechnol. (2015)33:985-989).

In certain embodiments, the guide molecule (capable of guiding Cas13 toa target locus) comprises (1) a guide sequence capable of hybridizing toa target locus and (2) a tracr mate or direct repeat sequence wherebythe direct repeat sequence is located upstream (i.e., 5′) from the guidesequence. In a particular embodiment the seed sequence (i.e. thesequence essential critical for recognition and/or hybridization to thesequence at the target locus) of the Cas13 guide sequence isapproximately within the first 10 nucleotides of the guide sequence. Inparticular embodiments, the Cas13 is FnCas13 and the seed sequence isapproximately within the first 5 nt on the 5′ end of the guide sequence.

In a particular embodiment the guide molecule comprises a guide sequencelinked to a direct repeat sequence, wherein the direct repeat sequencecomprises one or more stem loops or optimized secondary structures. Inparticular embodiments, the direct repeat has a minimum length of 16 ntsand a single stem loop. In further embodiments the direct repeat has alength longer than 16 nts, preferably more than 17 nts, and has morethan one stem loops or optimized secondary structures. In particularembodiments the guide molecule comprises or consists of the guidesequence linked to all or part of the natural direct repeat sequence. Atypical Type V Cas13 guide molecule comprises (in 3′ to 5′ direction): aguide sequence a first complimentary stretch (the “repeat”), a loop(which is typically 4 or 5 nucleotides long), a second complimentarystretch (the “anti-repeat” being complimentary to the repeat), and apoly A (often poly U in RNA) tail (terminator). In certain embodiments,the direct repeat sequence retains its natural architecture and forms asingle stem loop. In particular embodiments, certain aspects of theguide architecture can be modified, for example by addition,subtraction, or substitution of features, whereas certain other aspectsof guide architecture are maintained. Preferred locations for engineeredguide molecule modifications, including but not limited to insertions,deletions, and substitutions include guide termini and regions of theguide molecule that are exposed when complexed with the Cas13 proteinand/or target, for example the stemloop of the direct repeat sequence.

In particular embodiments, the stem comprises at least about 4 bpcomprising complementary X and Y sequences, although stems of more,e.g., 5, 6, 7, 8, 9, 10, 11 or 12 or fewer, e.g., 3, 2, base pairs arealso contemplated. Thus, for example X2-10 and Y2-10 (wherein X and Yrepresent any complementary set of nucleotides) may be contemplated. Inone aspect, the stem made of the X and Y nucleotides, together with theloop will form a complete hairpin in the overall secondary structure;and, this may be advantageous and the amount of base pairs can be anyamount that forms a complete hairpin. In one aspect, any complementaryX:Y basepairing sequence (e.g., as to length) is tolerated, so long asthe secondary structure of the entire guide molecule is preserved. Inone aspect, the loop that connects the stem made of X:Y basepairs can beany sequence of the same length (e.g., 4 or 5 nucleotides) or longerthat does not interrupt the overall secondary structure of the guidemolecule. In one aspect, the stemloop can further comprise, e.g. an MS2aptamer. In one aspect, the stem comprises about 5-7 bp comprisingcomplementary X and Y sequences, although stems of more or fewerbasepairs are also contemplated. In one aspect, non-Watson Crickbasepairing is contemplated, where such pairing otherwise generallypreserves the architecture of the stemloop at that position.

In particular embodiments the natural hairpin or stemloop structure ofthe guide molecule is extended or replaced by an extended stemloop. Ithas been demonstrated that extension of the stem can enhance theassembly of the guide molecule with the CRISPR-Cas protein (Chen et al.Cell. (2013); 155(7): 1479-1491). In particular embodiments the stem ofthe stemloop is extended by at least 1, 2, 3, 4, 5 or more complementarybasepairs (i.e. corresponding to the addition of 2,4, 6, 8, 10 or morenucleotides in the guide molecule). In particular embodiments these arelocated at the end of the stem, adjacent to the loop of the stemloop.

In particular embodiments, the susceptibility of the guide molecule toRNAses or to decreased expression can be reduced by slight modificationsof the sequence of the guide molecule which do not affect its function.For instance, in particular embodiments, premature termination oftranscription, such as premature transcription of U6 Pol-III, can beremoved by modifying a putative Pol-III terminator (4 consecutive U's)in the guide molecules sequence. Where such sequence modification isrequired in the stemloop of the guide molecule, it is preferably ensuredby a basepair flip.

In a preferred embodiment the direct repeat may be modified to compriseone or more protein-binding RNA aptamers. In a particular embodiment,one or more aptamers may be included such as part of optimized secondarystructure. Such aptamers may be capable of binding a bacteriophage coatprotein as detailed further herein.

In some embodiments, the guide molecule forms a duplex with a target DNAstrand comprising at least one target adenosine residues to be edited.Upon hybridization of the guide RNA molecule to the target DNA strand,the adenosine deaminase binds to the duplex and catalyzes deamination ofone or more target adenosine residues comprised within the DNA-RNAduplex.

A guide sequence, and hence a nucleic acid-targeting guide RNA may beselected to target any target nucleic acid sequence. The target sequencemay be DNA. The target sequence may be genomic DNA. The target sequencemay be mitochondrial DNA.

In certain embodiments, the target sequence should be associated with aPAM (protospacer adjacent motif) or PFS (protospacer flanking sequenceor site); that is, a short sequence recognized by the CRISPR complex.Depending on the nature of the CRISPR-Cas protein, the target sequenceshould be selected such that its complementary sequence in the DNAduplex (also referred to herein as the non-target sequence) is upstreamor downstream of the PAM. In the embodiments of the present inventionwhere the CRISPR-Cas protein is a Cas13 protein, the complementarysequence of the target sequence in a is downstream or 3′ of the PAM. Theprecise sequence and length requirements for the PAM differ depending onthe Cas13 protein used, but PAMs are typically 2-5 base pair sequencesadjacent the protospacer (that is, the target sequence). Examples of thenatural PAM sequences for different Cas13 orthologues are providedherein below and the skilled person will be able to identify further PAMsequences for use with a given Cas13 protein.

Further, engineering of the PAM Interacting (PI) domain may allowprogramming of PAM specificity, improve target site recognitionfidelity, and increase the versatility of the CRISPR-Cas protein, forexample as described for Cas9 in Kleinstiver B P et al. EngineeredCRISPR-Cas9 nucleases with altered PAM specificities. Nature. 2015 Jul.23; 523(7561):481-5. doi: 10.1038/nature14592. As further detailedherein, the skilled person will understand that Cas13 proteins may bemodified analogously.

In particular embodiments, the guide sequence is selected in order toensure optimal efficiency of the deaminase on the adenine to bedeaminated. The position of the adenine in the target strand relative tothe cleavage site of the Cas13 nickase may be taken into account. Inparticular embodiments it is of interest to ensure that the nickase willact in the vicinity of the adenine to be deaminated, on the non-targetstrand. For instance, in particular embodiments, the Cas13 nickase cutsthe non-targeting strand 17 nucleotides downstream of the PAM (e.g.AsCas13, LbCas13) or 18 nucleotides downstream of the PAM (e.g.FnCas13), and it can be of interest to design the guide that thecytosine which is to correspond to the adenine to be deaminated islocated in the guide sequence within 10 bp upstream or downstream of thenickase cleavage site in the sequence of the corresponding non-targetstrand.

In particular embodiment, the guide is an escorted guide. By “escorted”is meant that the Cas13 CRISPR-Cas system or complex or guide isdelivered to a selected time or place within a cell, so that activity ofthe Cas13 CRISPR-Cas system or complex or guide is spatially ortemporally controlled. For example, the activity and destination of theCas13 CRISPR-Cas system or complex or guide may be controlled by anescort RNA aptamer sequence that has binding affinity for an aptamerligand, such as a cell surface protein or other localized cellularcomponent. Alternatively, the escort aptamer may for example beresponsive to an aptamer effector on or in the cell, such as a transienteffector, such as an external energy source that is applied to the cellat a particular time.

The escorted Cas13 CRISPR-Cas systems or complexes have a guide moleculewith a functional structure designed to improve guide moleculestructure, architecture, stability, genetic expression, or anycombination thereof. Such a structure can include an aptamer.

Aptamers are biomolecules that can be designed or selected to bindtightly to other ligands, for example using a technique calledsystematic evolution of ligands by exponential enrichment (SELEX; TuerkC, Gold L: “Systematic evolution of ligands by exponential enrichment:RNA ligands to bacteriophage T4 DNA polymerase.” Science 1990,249:505-510). Nucleic acid aptamers can for example be selected frompools of random-sequence oligonucleotides, with high binding affinitiesand specificities for a wide range of biomedically relevant targets,suggesting a wide range of therapeutic utilities for aptamers (Keefe,Anthony D., Supriya Pai, and Andrew Ellington. “Aptamers astherapeutics.” Nature Reviews Drug Discovery 9.7 (2010): 537-550). Thesecharacteristics also suggest a wide range of uses for aptamers as drugdelivery vehicles (Levy-Nissenbaum, Etgar, et al. “Nanotechnology andaptamers: applications in drug delivery.” Trends in biotechnology 26.8(2008): 442-449; and, Hicke B J, Stephens A W. “Escort aptamers: adelivery service for diagnosis and therapy.” J Clin Invest 2000,106:923-928.). Aptamers may also be constructed that function asmolecular switches, responding to a que by changing properties, such asRNA aptamers that bind fluorophores to mimic the activity of greenfluorescent protein (Paige, Jeremy S., Karen Y. Wu, and Samie R.Jaffrey. “RNA mimics of green fluorescent protein.” Science 333.6042(2011): 642-646). It has also been suggested that aptamers may be usedas components of targeted siRNA therapeutic delivery systems, forexample targeting cell surface proteins (Zhou, Jiehua, and John J.Rossi. “Aptamer-targeted cell-specific RNA interference.” Silence 1.1(2010): 4).

Accordingly, in particular embodiments, the guide molecule is modified,e.g., by one or more aptamer(s) designed to improve guide moleculedelivery, including delivery across the cellular membrane, tointracellular compartments, or into the nucleus. Such a structure caninclude, either in addition to the one or more aptamer(s) or withoutsuch one or more aptamer(s), moiety(ies) so as to render the guidemolecule deliverable, inducible or responsive to a selected effector.The invention accordingly comprehends an guide molecule that responds tonormal or pathological physiological conditions, including withoutlimitation pH, hypoxia, O2 concentration, temperature, proteinconcentration, enzymatic concentration, lipid structure, light exposure,mechanical disruption (e.g. ultrasound waves), magnetic fields, electricfields, or electromagnetic radiation.

Light responsiveness of an inducible system may be achieved via theactivation and binding of cryptochrome-2 and CIB 1. Blue lightstimulation induces an activating conformational change incryptochrome-2, resulting in recruitment of its binding partner CIB1.This binding is fast and reversible, achieving saturation in <15 secfollowing pulsed stimulation and returning to baseline <15 min after theend of stimulation. These rapid binding kinetics result in a systemtemporally bound only by the speed of transcription/translation andtranscript/protein degradation, rather than uptake and clearance ofinducing agents. Crytochrome-2 activation is also highly sensitive,allowing for the use of low light intensity stimulation and mitigatingthe risks of phototoxicity. Further, in a context such as the intactmammalian brain, variable light intensity may be used to control thesize of a stimulated region, allowing for greater precision than vectordelivery alone may offer.

The invention contemplates energy sources such as electromagneticradiation, sound energy or thermal energy to induce the guide.Advantageously, the electromagnetic radiation is a component of visiblelight. In a preferred embodiment, the light is a blue light with awavelength of about 450 to about 495 nm. In an especially preferredembodiment, the wavelength is about 488 nm. In another preferredembodiment, the light stimulation is via pulses. The light power mayrange from about 0-9 mW/cm2. In a preferred embodiment, a stimulationparadigm of as low as 0.25 sec every 15 sec should result in maximalactivation.

The chemical or energy sensitive guide may undergo a conformationalchange upon induction by the binding of a chemical source or by theenergy allowing it act as a guide and have the Cas13 CRISPR-Cas systemor complex function. The invention can involve applying the chemicalsource or energy so as to have the guide function and the Cas13CRISPR-Cas system or complex function; and optionally furtherdetermining that the expression of the genomic locus is altered.

There are several different designs of this chemical induciblesystem: 1. ABI-PYL based system inducible by Abscisic Acid (ABA) (see,e.g., stke.sciencemag.org/cgi/content/abstract/sigtrans;4/164/r52), 2.FKBP-FRB based system inducible by rapamycin (or related chemicals basedon rapamycin) (see, e.g.,www.nature.com/nmeth/journal/v2/n6/full/nmeth763.html), 3. GID1-GAIbased system inducible by Gibberellin (GA) (see, e.g.,www.nature.com/nchembio/journal/v8/n5/full/nchembio.922.html).

A chemical inducible system can be an estrogen receptor (ER) basedsystem inducible by 4-hydroxytamoxifen (4OHT) (see, e.g.,www.pnas.org/content/104/3/1027.abstract). A mutated ligand-bindingdomain of the estrogen receptor called ERT2 translocates into thenucleus of cells upon binding of 4-hydroxytamoxifen. In furtherembodiments of the invention any naturally occurring or engineeredderivative of any nuclear receptor, thyroid hormone receptor, retinoicacid receptor, estrogren receptor, estrogen-related receptor,glucocorticoid receptor, progesterone receptor, androgen receptor may beused in inducible systems analogous to the ER based inducible system.

Another inducible system is based on the design using Transient receptorpotential (TRP) ion channel based system inducible by energy, heat orradio-wave (see, e.g., www.sciencemag.org/content/336/6081/604). TheseTRP family proteins respond to different stimuli, including light andheat. When this protein is activated by light or heat, the ion channelwill open and allow the entering of ions such as calcium into the plasmamembrane. This influx of ions will bind to intracellular ion interactingpartners linked to a polypeptide including the guide and the othercomponents of the Cas13 CRISPR-Cas complex or system, and the bindingwill induce the change of sub-cellular localization of the polypeptide,leading to the entire polypeptide entering the nucleus of cells. Onceinside the nucleus, the guide protein and the other components of theCas13 CRISPR-Cas complex will be active and modulating target geneexpression in cells.

While light activation may be an advantageous embodiment, sometimes itmay be disadvantageous especially for in vivo applications in which thelight may not penetrate the skin or other organs. In this instance,other methods of energy activation are contemplated, in particular,electric field energy and/or ultrasound which have a similar effect.

Electric field energy is preferably administered substantially asdescribed in the art, using one or more electric pulses of from about 1Volt/cm to about 10 kVolts/cm under in vivo conditions. Instead of or inaddition to the pulses, the electric field may be delivered in acontinuous manner. The electric pulse may be applied for between 1 μsand 500 milliseconds, preferably between 1 μs and 100 milliseconds. Theelectric field may be applied continuously or in a pulsed manner for 5about minutes.

As used herein, ‘electric field energy’ is the electrical energy towhich a cell is exposed. Preferably the electric field has a strength offrom about 1 Volt/cm to about 10 kVolts/cm or more under in vivoconditions (see WO97/49450).

As used herein, the term “electric field” includes one or more pulses atvariable capacitance and voltage and including exponential and/or squarewave and/or modulated wave and/or modulated square wave forms.References to electric fields and electricity should be taken to includereference the presence of an electric potential difference in theenvironment of a cell. Such an environment may be set up by way ofstatic electricity, alternating current (AC), direct current (DC), etc,as known in the art. The electric field may be uniform, non-uniform orotherwise, and may vary in strength and/or direction in a time dependentmanner.

Single or multiple applications of electric field, as well as single ormultiple applications of ultrasound are also possible, in any order andin any combination. The ultrasound and/or the electric field may bedelivered as single or multiple continuous applications, or as pulses(pulsatile delivery).

Electroporation has been used in both in vitro and in vivo procedures tointroduce foreign material into living cells. With in vitroapplications, a sample of live cells is first mixed with the agent ofinterest and placed between electrodes such as parallel plates. Then,the electrodes apply an electrical field to the cell/implant mixture.Examples of systems that perform in vitro electroporation include theElectro Cell Manipulator ECM600 product, and the Electro Square PoratorT820, both made by the BTX Division of Genetronics, Inc (see U.S. Pat.No 5,869,326).

The known electroporation techniques (both in vitro and in vivo)function by applying a brief high voltage pulse to electrodes positionedaround the treatment region. The electric field generated between theelectrodes causes the cell membranes to temporarily become porous,whereupon molecules of the agent of interest enter the cells. In knownelectroporation applications, this electric field comprises a singlesquare wave pulse on the order of 1000 V/cm, of about 100 .mu.sduration. Such a pulse may be generated, for example, in knownapplications of the Electro Square Porator T820.

Preferably, the electric field has a strength of from about 1 V/cm toabout 10 kV/cm under in vitro conditions. Thus, the electric field mayhave a strength of 1 V/cm, 2 V/cm, 3 V/cm, 4 V/cm, 5 V/cm, 6 V/cm, 7V/cm, 8 V/cm, 9 V/cm, 10 V/cm, 20 V/cm, 50 V/cm, 100 V/cm, 200 V/cm, 300V/cm, 400 V/cm, 500 V/cm, 600 V/cm, 700 V/cm, 800 V/cm, 900 V/cm, 1kV/cm, 2 kV/cm, 5 kV/cm, 10 kV/cm, 20 kV/cm, 50 kV/cm or more. Morepreferably from about 0.5 kV/cm to about 4.0 kV/cm under in vitroconditions. Preferably the electric field has a strength of from about 1V/cm to about 10 kV/cm under in vivo conditions. However, the electricfield strengths may be lowered where the number of pulses delivered tothe target site are increased. Thus, pulsatile delivery of electricfields at lower field strengths is envisaged.

Preferably the application of the electric field is in the form ofmultiple pulses such as double pulses of the same strength andcapacitance or sequential pulses of varying strength and/or capacitance.As used herein, the term “pulse” includes one or more electric pulses atvariable capacitance and voltage and including exponential and/or squarewave and/or modulated wave/square wave forms.

Preferably the electric pulse is delivered as a waveform selected froman exponential wave form, a square wave form, a modulated wave form anda modulated square wave form.

A preferred embodiment employs direct current at low voltage. Thus,Applicants disclose the use of an electric field which is applied to thecell, tissue or tissue mass at a field strength of between 1 V/cm and 20V/cm, for a period of 100 milliseconds or more, preferably 15 minutes ormore.

Ultrasound is advantageously administered at a power level of from about0.05 W/cm2 to about 100 W/cm2. Diagnostic or therapeutic ultrasound maybe used, or combinations thereof.

As used herein, the term “ultrasound” refers to a form of energy whichconsists of mechanical vibrations the frequencies of which are so highthey are above the range of human hearing. Lower frequency limit of theultrasonic spectrum may generally be taken as about 20 kHz. Mostdiagnostic applications of ultrasound employ frequencies in the range 1and 15 MHz′ (From Ultrasonics in Clinical Diagnosis, P. N. T. Wells,ed., 2nd. Edition, Publ. Churchill Livingstone [Edinburgh, London & NY,1977]).

Ultrasound has been used in both diagnostic and therapeuticapplications. When used as a diagnostic tool (“diagnostic ultrasound”),ultrasound is typically used in an energy density range of up to about100 mW/cm2 (FDA recommendation), although energy densities of up to 750mW/cm2 have been used. In physiotherapy, ultrasound is typically used asan energy source in a range up to about 3 to 4 W/cm2 (WHOrecommendation). In other therapeutic applications, higher intensitiesof ultrasound may be employed, for example, HIFU at 100 W/cm up to 1kW/cm2 (or even higher) for short periods of time. The term “ultrasound”as used in this specification is intended to encompass diagnostic,therapeutic and focused ultrasound.

Focused ultrasound (FUS) allows thermal energy to be delivered withoutan invasive probe (see Morocz et al 1998 Journal of Magnetic ResonanceImaging Vol. 8, No. 1, pp. 136-142. Another form of focused ultrasoundis high intensity focused ultrasound (HIFU) which is reviewed byMoussatov et al in Ultrasonics (1998) Vol. 36, No. 8, pp. 893-900 andTranHuuHue et al in Acustica (1997) Vol. 83, No. 6, pp. 1103-1106.

Preferably, a combination of diagnostic ultrasound and a therapeuticultrasound is employed. This combination is not intended to be limiting,however, and the skilled reader will appreciate that any variety ofcombinations of ultrasound may be used. Additionally, the energydensity, frequency of ultrasound, and period of exposure may be varied.

Preferably the exposure to an ultrasound energy source is at a powerdensity of from about 0.05 to about 100 Wcm−2. Even more preferably, theexposure to an ultrasound energy source is at a power density of fromabout 1 to about 15 Wcm−2.

Preferably the exposure to an ultrasound energy source is at a frequencyof from about 0.015 to about 10.0 MHz. More preferably the exposure toan ultrasound energy source is at a frequency of from about 0.02 toabout 5.0 MHz or about 6.0 MHz. Most preferably, the ultrasound isapplied at a frequency of 3 MHz.

Preferably the exposure is for periods of from about 10 milliseconds toabout 60 minutes. Preferably the exposure is for periods of from about 1second to about 5 minutes. More preferably, the ultrasound is appliedfor about 2 minutes. Depending on the particular target cell to bedisrupted, however, the exposure may be for a longer duration, forexample, for 15 minutes.

Advantageously, the target tissue is exposed to an ultrasound energysource at an acoustic power density of from about 0.05 Wcm−2 to about 10Wcm−2 with a frequency ranging from about 0.015 to about 10 MHz (see WO98/52609). However, alternatives are also possible, for example,exposure to an ultrasound energy source at an acoustic power density ofabove 100 Wcm−2, but for reduced periods of time, for example, 1000Wcm−2 for periods in the millisecond range or less.

Preferably the application of the ultrasound is in the form of multiplepulses; thus, both continuous wave and pulsed wave (pulsatile deliveryof ultrasound) may be employed in any combination. For example,continuous wave ultrasound may be applied, followed by pulsed waveultrasound, or vice versa. This may be repeated any number of times, inany order and combination. The pulsed wave ultrasound may be appliedagainst a background of continuous wave ultrasound, and any number ofpulses may be used in any number of groups.

Preferably, the ultrasound may comprise pulsed wave ultrasound. In ahighly preferred embodiment, the ultrasound is applied at a powerdensity of 0.7 Wcm−2 or 1.25 Wcm−2 as a continuous wave. Higher powerdensities may be employed if pulsed wave ultrasound is used.

Use of ultrasound is advantageous as, like light, it may be focusedaccurately on a target. Moreover, ultrasound is advantageous as it maybe focused more deeply into tissues unlike light. It is therefore bettersuited to whole-tissue penetration (such as but not limited to a lobe ofthe liver) or whole organ (such as but not limited to the entire liveror an entire muscle, such as the heart) therapy. Another importantadvantage is that ultrasound is a non-invasive stimulus which is used ina wide variety of diagnostic and therapeutic applications. By way ofexample, ultrasound is well known in medical imaging techniques and,additionally, in orthopedic therapy. Furthermore, instruments suitablefor the application of ultrasound to a subject vertebrate are widelyavailable and their use is well known in the art.

In particular embodiments, the guide molecule is modified by a secondarystructure to increase the specificity of the CRISPR-Cas system and thesecondary structure can protect against exonuclease activity and allowfor 5′ additions to the guide sequence also referred to herein as aprotected guide molecule.

In one aspect, the invention provides for hybridizing a “protector RNA”to a sequence of the guide molecule, wherein the “protector RNA” is anRNA strand complementary to the 3′ end of the guide molecule to therebygenerate a partially double-stranded guide RNA. In an embodiment of theinvention, protecting mismatched bases (i.e. the bases of the guidemolecule which do not form part of the guide sequence) with a perfectlycomplementary protector sequence decreases the likelihood of target DNAbinding to the mismatched basepairs at the 3′ end. In particularembodiments of the invention, additional sequences comprising anextended length may also be present within the guide molecule such thatthe guide comprises a protector sequence within the guide molecule. This“protector sequence” ensures that the guide molecule comprises a“protected sequence” in addition to an “exposed sequence” (comprisingthe part of the guide sequence hybridizing to the target sequence). Inparticular embodiments, the guide molecule is modified by the presenceof the protector guide to comprise a secondary structure such as ahairpin. Advantageously there are three or four to thirty or more, e.g.,about 10 or more, contiguous base pairs having complementarity to theprotected sequence, the guide sequence or both. It is advantageous thatthe protected portion does not impede thermodynamics of the CRISPR-Cassystem interacting with its target. By providing such an extensionincluding a partially double stranded guide molecule, the guide moleculeis considered protected and results in improved specific binding of theCRISPR-Cas complex, while maintaining specific activity.

In particular embodiments, use is made of a truncated guide (tru-guide),i.e. a guide molecule which comprises a guide sequence which istruncated in length with respect to the canonical guide sequence length.As described by Nowak et al. (Nucleic Acids Res (2016) 44 (20):9555-9564), such guides may allow catalytically active CRISPR-Cas enzymeto bind its target without cleaving the target DNA. In particularembodiments, a truncated guide is used which allows the binding of thetarget but retains only nickase activity of the CRISPR-Cas enzyme.

Crispr-Cas Enzyme

In its unmodified form, a CRISPR-Cas protein is a catalytically activeprotein. This implies that upon formation of a nucleic acid-targetingcomplex (comprising a guide RNA hybridized to a target sequence one orboth DNA strands in or near (e.g., within 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,20, 50, or more base pairs from) the target sequence is modified (e.g.cleaved). As used herein the term “sequence(s) associated with a targetlocus of interest” refers to sequences near the vicinity of the targetsequence (e.g. within 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 50, or morebase pairs from the target sequence, wherein the target sequence iscomprised within a target locus of interest). The unmodifiedcatalytically active Cas13 protein generates a staggered cut, wherebythe cut sites are typically within the target sequence. Moreparticularly, the staggered cut is typically 13-23 nucleotides distal tothe PAM. In particular embodiments, the cut on the non-target strand is17 nucleotides downstream of the PAM (i.e. between nucleotide 17 and 18downstream of the PAM), while the cut on the target strand (i.e. strandhybridizing with the guide sequence) occurs a further 4 nucleotidesfurther from the sequence complementary to the PAM (this is 21nucleotides upstream of the complement of the PAM on the 3′ strand orbetween nucleotide 21 and 22 upstream of the complement of the PAM).

In the methods according to the present invention, the CRISPR-Casprotein is preferably mutated with respect to a corresponding wild-typeenzyme such that the mutated CRISPR-Cas protein lacks the ability tocleave one or both DNA strands of a target locus containing a targetsequence. In particular embodiments, one or more catalytic domains ofthe Cas13 protein are mutated to produce a mutated Cas protein whichcleaves only one DNA strand of a target sequence.

In particular embodiments, the CRISPR-Cas protein may be mutated withrespect to a corresponding wild-type enzyme such that the mutatedCRISPR-Cas protein lacks substantially all DNA cleavage activity. Insome embodiments, a CRISPR-Cas protein may be considered tosubstantially lack all DNA and/or RNA cleavage activity when thecleavage activity of the mutated enzyme is about no more than 25%, 10%,5%, 1%, 0.1%, 0.01%, or less of the nucleic acid cleavage activity ofthe non-mutated form of the enzyme; an example can be when the nucleicacid cleavage activity of the mutated form is nil or negligible ascompared with the non-mutated form.

In certain embodiments of the methods provided herein the CRISPR-Casprotein is a mutated CRISPR-Cas protein which cleaves only one DNAstrand, i.e. a nickase. More particularly, in the context of the presentinvention, the nickase ensures cleavage within the non-target sequence,i.e. the sequence which is on the opposite DNA strand of the targetsequence and which is 3′ of the PAM sequence. By means of furtherguidance, and without limitation, an arginine-to-alanine substitution(R1226A) in the Nuc domain of Cas13 from Acidaminococcus sp. convertsCas13 from a nuclease that cleaves both strands to a nickase (cleaves asingle strand). It will be understood by the skilled person that wherethe enzyme is not AsCas13, a mutation may be made at a residue in acorresponding position. In particular embodiments, the Cas13 is FnCas13and the mutation is at the arginine at position R1218. In particularembodiments, the Cas13 is LbCas13 and the mutation is at the arginine atposition R1138. In particular embodiments, the Cas13 is MbCas13 and themutation is at the arginine at position R1293.

In certain embodiments of the methods provided herein the CRISPR-Casprotein has reduced or no catalytic activity. Where the CRISPR-Casprotein is a Cas13 protein, the mutations may include but are notlimited to one or more mutations in the catalytic RuvC-like domain, suchas D908A or E993A with reference to the positions in AsCas13.

In some embodiments, a CRISPR-Cas protein is considered to substantiallylack all DNA cleavage activity when the DNA cleavage activity of themutated enzyme is about no more than 25%, 10%, 5%, 1%, 0.1%, 0.01%, orless of the DNA cleavage activity of the non-mutated form of the enzyme;an example can be when the DNA cleavage activity of the mutated form isnil or negligible as compared with the non-mutated form. In theseembodiments, the CRISPR-Cas protein is used as a generic DNA bindingprotein. The mutations may be artificially introduced mutations or gain-or loss-of-function mutations.

In addition to the mutations described above, the CRISPR-Cas protein maybe additionally modified. As used herein, the term “modified” withregard to a CRISPR-Cas protein generally refers to a CRISPR-Cas proteinhaving one or more modifications or mutations (including pointmutations, truncations, insertions, deletions, chimeras, fusionproteins, etc.) compared to the wild type Cas protein from which it isderived. By derived is meant that the derived enzyme is largely based,in the sense of having a high degree of sequence homology with, awildtype enzyme, but that it has been mutated (modified) in some way asknown in the art or as described herein.

The additional modifications of the CRISPR-Cas protein may or may notcause an altered functionality. By means of example, and in particularwith reference to CRISPR-Cas protein, modifications which do not resultin an altered functionality include for instance codon optimization forexpression into a particular host, or providing the nuclease with aparticular marker (e.g. for visualization). Modifications with mayresult in altered functionality may also include mutations, includingpoint mutations, insertions, deletions, truncations (including splitnucleases), etc. Fusion proteins may without limitation include forinstance fusions with heterologous domains or functional domains (e.g.localization signals, catalytic domains, etc.). In certain embodiments,various different modifications may be combined (e.g. a mutated nucleasewhich is catalytically inactive and which further is fused to afunctional domain, such as for instance to induce DNA methylation oranother nucleic acid modification, such as including without limitationa break (e.g. by a different nuclease (domain)), a mutation, a deletion,an insertion, a replacement, a ligation, a digestion, a break or arecombination). As used herein, “altered functionality” includes withoutlimitation an altered specificity (e.g. altered target recognition,increased (e.g. “enhanced” Cas proteins) or decreased specificity, oraltered PAM recognition), altered activity (e.g. increased or decreasedcatalytic activity, including catalytically inactive nucleases ornickases), and/or altered stability (e.g. fusions with destalilizationdomains). Suitable heterologous domains include without limitation anuclease, a ligase, a repair protein, a methyltransferase, (viral)integrase, a recombinase, a transposase, an argonaute, a cytidinedeaminase, a retron, a group II intron, a phosphatase, a phosphorylase,a sulpfurylase, a kinase, a polymerase, an exonuclease, etc. Examples ofall these modifications are known in the art. It will be understood thata “modified” nuclease as referred to herein, and in particular a“modified” Cas or “modified” CRISPR-Cas system or complex preferablystill has the capacity to interact with or bind to the polynucleic acid(e.g. in complex with the guide molecule). Such modified Cas protein canbe combined with the deaminase protein or active domain thereof asdescribed herein.

In certain embodiments, CRISPR-Cas protein may comprise one or moremodifications resulting in enhanced activity and/or specificity, such asincluding mutating residues that stabilize the targeted or non-targetedstrand (e.g. eCas9; “Rationally engineered Cas9 nucleases with improvedspecificity”, Slaymaker et al. (2016), Science, 351(6268):84-88,incorporated herewith in its entirety by reference). In certainembodiments, the altered or modified activity of the engineered CRISPRprotein comprises increased targeting efficiency or decreased off-targetbinding. In certain embodiments, the altered activity of the engineeredCRISPR protein comprises modified cleavage activity. In certainembodiments, the altered activity comprises increased cleavage activityas to the target polynucleotide loci. In certain embodiments, thealtered activity comprises decreased cleavage activity as to the targetpolynucleotide loci. In certain embodiments, the altered activitycomprises decreased cleavage activity as to off-target polynucleotideloci. In certain embodiments, the altered or modified activity of themodified nuclease comprises altered helicase kinetics. In certainembodiments, the modified nuclease comprises a modification that altersassociation of the protein with the nucleic acid molecule comprising RNA(in the case of a Cas protein), or a strand of the target polynucleotideloci, or a strand of off-target polynucleotide loci. In an aspect of theinvention, the engineered CRISPR protein comprises a modification thatalters formation of the CRISPR complex. In certain embodiments, thealtered activity comprises increased cleavage activity as to off-targetpolynucleotide loci. Accordingly, in certain embodiments, there isincreased specificity for target polynucleotide loci as compared tooff-target polynucleotide loci. In other embodiments, there is reducedspecificity for target polynucleotide loci as compared to off-targetpolynucleotide loci. In certain embodiments, the mutations result indecreased off-target effects (e.g. cleavage or binding properties,activity, or kinetics), such as in case for Cas proteins for instanceresulting in a lower tolerance for mismatches between target and guideRNA. Other mutations may lead to increased off-target effects (e.g.cleavage or binding properties, activity, or kinetics). Other mutationsmay lead to increased or decreased on-target effects (e.g. cleavage orbinding properties, activity, or kinetics). In certain embodiments, themutations result in altered (e.g. increased or decreased) helicaseactivity, association or formation of the functional nuclease complex(e.g. CRISPR-Cas complex). In certain embodiments, as described above,the mutations result in an altered PAM recognition, i.e. a different PAMmay be (in addition or in the alternative) be recognized, compared tothe unmodified Cas protein. Particularly preferred mutations includepositively charged residues and/or (evolutionary) conserved residues,such as conserved positively charged residues, in order to enhancespecificity. In certain embodiments, such residues may be mutated touncharged residues, such as alanine.

Base Editing

In some embodiments, the systems may be used for base editing. Forexample, the systems disclosed herein comprise a targeting component anda base editing component. The targeting component functions tospecifically target the base editing component to a target nucleotidesequence in which one or more nucleotides are to be edited. In someexamples, the target component may be a catalytically inactive Cas13effector protein (e.g., dCas13). The base editing component may thencatalyze a chemical reaction to convert a first nucleotide in the targetsequence to a second nucleotide. For example, the base editor maycatalyze conversion of an adenine such that it is read as guanine by acell's transcription or translation machinery, or vice versa. Likewise,the base editing component may catalyze conversion of cytidine to auracil, or vice versa. In certain example embodiments, the base editormay be derived by starting with a known base editor, such as an adeninedeaminase or cytidine deaminase, and modified using methods such asdirected evolution to derive new functionalities. Directed evolutiontechniques are known in the art and may include those described in WO2015/184016 “High-Throughput Assembly of Genetic Permuatations.”

In certain example embodiments, the binding component may be aDNA-binding protein of functional domain thereof, or a RNA-bindingdomain or functional domain thereof. The binding component may bind asequence, motif, or structural feature of the at or adjacent to thetarget locus. A structural feature may include hairpins, tetraloops, orother secondary structural features of a nucleic acid. As used herein“adjacent” means within a distance and/or orientation of the targetlocus in which the adenosine deaminase can complete its base editingfunction. In certain example embodiments, the binding component thereofmay bind a sequence, motif, or structural sequence of a guide molecule.The guide molecule comprises a sequence that hybridizes to the targetloci of interest and thereby directs the adenosine deaminase to thetarget loci.

Adenosine Deaminase

In certain example embodiments, base editing component is an adenosinedeaminase. The adenosine deaminase is recruited to the target locus bythe targeting component. In certain example embodiments, the adenosinedeaminase is linked to a RNA-binding protein or functional fragmentthereof, or a DNA-binding protein or functional fragment thereof.

The term “adenosine deaminase” or “adenosine deaminase protein” as usedherein refers to a protein, a polypeptide, or one or more functionaldomain(s) of a protein or a polypeptide that is capable of catalyzing ahydrolytic deamination reaction that converts an adenine (or an adeninemoiety of a molecule) to a hypoxanthine (or a hypoxanthine moiety of amolecule), as shown below. In some embodiments, the adenine-containingmolecule is an adenosine (A), and the hypoxanthine-containing moleculeis an inosine (I). The adenine-containing molecule can bedeoxyribonucleic acid (DNA) or ribonucleic acid (RNA).

According to the present disclosure, adenosine deaminases that can beused in connection with the present disclosure include, but are notlimited to, members of the enzyme family known as adenosine deaminasesthat act on RNA (ADARs), members of the enzyme family known as adenosinedeaminases that act on tRNA (ADATs), and other adenosine deaminasedomain-containing (ADAD) family members. According to the presentdisclosure, the adenosine deaminase is capable of targeting adenine in aRNA/DNA and RNA duplexes. Indeed, Zheng et al. (Nucleic Acids Res. 2017,45(6): 3369-3377) demonstrate that ADARs can carry out adenosine toinosine editing reactions on RNA/DNA and RNA/RNA duplexes. In particularembodiments, the adenosine deaminase has been modified to increase itsability to edit DNA in a RNA/DNAn RNA duplex as detailed herein below.

In some embodiments, the adenosine deaminase is derived from one or moremetazoa species, including but not limited to, mammals, birds, frogs,squids, fish, flies and worms. In some embodiments, the adenosinedeaminase is a human, squid or Drosophila adenosine deaminase.

In some embodiments, the adenosine deaminase is a human ADAR, includinghADAR1, hADAR2, hADAR3. In some embodiments, the adenosine deaminase isa Caenorhabditis elegans ADAR protein, including ADR-1 and ADR-2. Insome embodiments, the adenosine deaminase is a Drosophila ADAR protein,including dAdar. In some embodiments, the adenosine deaminase is a squidLoligo pealeii ADAR protein, including sqADAR2a and sqADAR2b. In someembodiments, the adenosine deaminase is a human ADAT protein. In someembodiments, the adenosine deaminase is a Drosophila ADAT protein. Insome embodiments, the adenosine deaminase is a human ADAD protein,including TENR (hADAD1) and TENRL (hADAD2).

In some embodiments, the adenosine deaminase protein recognizes andconverts one or more target adenosine residue(s) in a double-strandednucleic acid substrate into inosine residues (s). In some embodiments,the double-stranded nucleic acid substrate is a RNA-DNA hybrid duplex.In some embodiments, the adenosine deaminase protein recognizes abinding window on the double-stranded substrate. In some embodiments,the binding window contains at least one target adenosine residue(s). Insome embodiments, the binding window is in the range of about 3 bp toabout 100 bp. In some embodiments, the binding window is in the range ofabout 5 bp to about 50 bp. In some embodiments, the binding window is inthe range of about 10 bp to about 30 bp. In some embodiments, thebinding window is about 1 bp, 2 bp, 3 bp, 5 bp, 7 bp, 10 bp, 15 bp, 20bp, 25 bp, 30 bp, 40 bp, 45 bp, 50 bp, 55 bp, 60 bp, 65 bp, 70 bp, 75bp, 80 bp, 85 bp, 90 bp, 95 bp, or 100 bp.

In some embodiments, the adenosine deaminase protein comprises one ormore deaminase domains. Not intended to be bound by theory, it iscontemplated that the deaminase domain functions to recognize andconvert one or more target adenosine (A) residue(s) contained in adouble-stranded nucleic acid substrate into inosine (I) residues (s). Insome embodiments, the deaminase domain comprises an active center. Insome embodiments, the active center comprises a zinc ion. In someembodiments, during the A-to-I editing process, base pairing at thetarget adenosine residue is disrupted, and the target adenosine residueis “flipped” out of the double helix to become accessible by theadenosine deaminase. In some embodiments, amino acid residues in or nearthe active center interact with one or more nucleotide(s) 5′ to a targetadenosine residue. In some embodiments, amino acid residues in or nearthe active center interact with one or more nucleotide(s) 3′ to a targetadenosine residue. In some embodiments, amino acid residues in or nearthe active center further interact with the nucleotide complementary tothe target adenosine residue on the opposite strand. In someembodiments, the amino acid residues form hydrogen bonds with the 2′hydroxyl group of the nucleotides.

In some embodiments, the adenosine deaminase comprises human ADAR2 fullprotein (hADAR2) or the deaminase domain thereof (hADAR2-D). In someembodiments, the adenosine deaminase is an ADAR family member that ishomologous to hADAR2 or hADAR2-D.

Particularly, in some embodiments, the homologous ADAR protein is humanADAR1 (hADAR1) or the deaminase domain thereof (hADAR1-D). In someembodiments, glycine 1007 of hADAR1-D corresponds to glycine 487hADAR2-D, and glutamic Acid 1008 of hADAR1-D corresponds to glutamicacid 488 of hADAR2-D.

In some embodiments, the adenosine deaminase comprises the wild-typeamino acid sequence of hADAR2-D. In some embodiments, the adenosinedeaminase comprises one or more mutations in the hADAR2-D sequence, suchthat the editing efficiency, and/or substrate editing preference ofhADAR2-D is changed according to specific needs.

Certain mutations of hADAR1 and hADAR2 proteins have been described inKuttan et al., Proc Natl Acad Sci U S A. (2012) 109(48):E3295-304; Wantet al. ACS Chem Biol. (2015) 10(11):2512-9; and Zheng et al. NucleicAcids Res. (2017) 45(6):3369-337, each of which is incorporated hereinby reference in its entirety.

In some embodiments, the adenosine deaminase comprises a mutation atglycine336 of the hADAR2-D amino acid sequence, or a correspondingposition in a homologous ADAR protein. In some embodiments, the glycineresidue at position 336 is replaced by an aspartic acid residue (G336D).

In some embodiments, the adenosine deaminase comprises a mutation atGlycine487 of the hADAR2-D amino acid sequence, or a correspondingposition in a homologous ADAR protein. In some embodiments, the glycineresidue at position 487 is replaced by a non-polar amino acid residuewith relatively small side chains. For example, in some embodiments, theglycine residue at position 487 is replaced by an alanine residue(G487A). In some embodiments, the glycine residue at position 487 isreplaced by a valine residue (G487V). In some embodiments, the glycineresidue at position 487 is replaced by an amino acid residue withrelatively large side chains. In some embodiments, the glycine residueat position 487 is replaced by a arginine residue (G487R). In someembodiments, the glycine residue at position 487 is replaced by a lysineresidue (G487K). In some embodiments, the glycine residue at position487 is replaced by a tryptophan residue (G487W). In some embodiments,the glycine residue at position 487 is replaced by a tyrosine residue(G487Y).

In some embodiments, the adenosine deaminase comprises a mutation atglutamic acid488 of the hADAR2-D amino acid sequence, or a correspondingposition in a homologous ADAR protein. In some embodiments, the glutamicacid residue at position 488 is replaced by a glutamine residue (E488Q).In some embodiments, the glutamic acid residue at position 488 isreplaced by a histidine residue (E488H). In some embodiments, theglutamic acid residue at position 488 is replace by an arginine residue(E488R). In some embodiments, the glutamic acid residue at position 488is replace by a lysine residue (E488K). In some embodiments, theglutamic acid residue at position 488 is replace by an asparagineresidue (E488N). In some embodiments, the glutamic acid residue atposition 488 is replace by an alanine residue (E488A). In someembodiments, the glutamic acid residue at position 488 is replace by aMethionine residue (E488M). In some embodiments, the glutamic acidresidue at position 488 is replace by a serine residue (E488S). In someembodiments, the glutamic acid residue at position 488 is replace by aphenylalanine residue (E488F). In some embodiments, the glutamic acidresidue at position 488 is replace by a lysine residue (E488L). In someembodiments, the glutamic acid residue at position 488 is replace by atryptophan residue (E488W).

In some embodiments, the adenosine deaminase comprises a mutation atthreonine490 of the hADAR2-D amino acid sequence, or a correspondingposition in a homologous ADAR protein. In some embodiments, thethreonine residue at position 490 is replaced by a cysteine residue(T490C). In some embodiments, the threonine residue at position 490 isreplaced by a serine residue (T490S). In some embodiments, the threonineresidue at position 490 is replaced by an alanine residue (T490A). Insome embodiments, the threonine residue at position 490 is replaced by aphenylalanine residue (T490F). In some embodiments, the threonineresidue at position 490 is replaced by a tyrosine residue (T490Y). Insome embodiments, the threonine residue at position 490 is replaced by aserine residue (T490R). In some embodiments, the threonine residue atposition 490 is replaced by an alanine residue (T490K). In someembodiments, the threonine residue at position 490 is replaced by aphenylalanine residue (T490P). In some embodiments, the threonineresidue at position 490 is replaced by a tyrosine residue (T490E).

In some embodiments, the adenosine deaminase comprises a mutation atvaline493 of the hADAR2-D amino acid sequence, or a correspondingposition in a homologous ADAR protein. In some embodiments, the valineresidue at position 493 is replaced by an alanine residue (V493A). Insome embodiments, the valine residue at position 493 is replaced by aserine residue (V493S). In some embodiments, the valine residue atposition 493 is replaced by a threonine residue (V493T). In someembodiments, the valine residue at position 493 is replaced by anarginine residue (V493R). In some embodiments, the valine residue atposition 493 is replaced by an aspartic acid residue (V493D). In someembodiments, the valine residue at position 493 is replaced by a prolineresidue (V493P). In some embodiments, the valine residue at position 493is replaced by a glycine residue (V493G).

In some embodiments, the adenosine deaminase comprises a mutation atalanine589 of the hADAR2-D amino acid sequence, or a correspondingposition in a homologous ADAR protein. In some embodiments, the alanineresidue at position 589 is replaced by a valine residue (A589V).

In some embodiments, the adenosine deaminase comprises a mutation atasparagine597 of the hADAR2-D amino acid sequence, or a correspondingposition in a homologous ADAR protein. In some embodiments, theasparagine residue at position 597 is replaced by a lysine residue(N597K). In some embodiments, the adenosine deaminase comprises amutation at position 597 of the amino acid sequence, which has anasparagine residue in the wild type sequence. In some embodiments, theasparagine residue at position 597 is replaced by an arginine residue(N597R). In some embodiments, the adenosine deaminase comprises amutation at position 597 of the amino acid sequence, which has anasparagine residue in the wild type sequence. In some embodiments, theasparagine residue at position 597 is replaced by an alanine residue(N597A). In some embodiments, the adenosine deaminase comprises amutation at position 597 of the amino acid sequence, which has anasparagine residue in the wild type sequence. In some embodiments, theasparagine residue at position 597 is replaced by a glutamic acidresidue (N597E). In some embodiments, the adenosine deaminase comprisesa mutation at position 597 of the amino acid sequence, which has anasparagine residue in the wild type sequence. In some embodiments, theasparagine residue at position 597 is replaced by a histidine residue(N597H). In some embodiments, the adenosine deaminase comprises amutation at position 597 of the amino acid sequence, which has anasparagine residue in the wild type sequence. In some embodiments, theasparagine residue at position 597 is replaced by a glycine residue(N597G). In some embodiments, the adenosine deaminase comprises amutation at position 597 of the amino acid sequence, which has anasparagine residue in the wild type sequence. In some embodiments, theasparagine residue at position 597 is replaced by a tyrosine residue(N597Y). In some embodiments, the asparagine residue at position 597 isreplaced by a phenylalanine residue (N597F).

In some embodiments, the adenosine deaminase comprises a mutation atserine599 of the hADAR2-D amino acid sequence, or a correspondingposition in a homologous ADAR protein. In some embodiments, the serineresidue at position 599 is replaced by a threonine residue (S599T).

In some embodiments, the adenosine deaminase comprises a mutation atasparagine613 of the hADAR2-D amino acid sequence, or a correspondingposition in a homologous ADAR protein. In some embodiments, theasparagine residue at position 613 is replaced by a lysine residue(N613K). In some embodiments, the adenosine deaminase comprises amutation at position 613 of the amino acid sequence, which has anasparagine residue in the wild type sequence. In some embodiments, theasparagine residue at position 613 is replaced by an arginine residue(N613R). In some embodiments, the adenosine deaminase comprises amutation at position 613 of the amino acid sequence, which has anasparagine residue in the wild type sequence. In some embodiments, theasparagine residue at position 613 is replaced by an alanine residue(N613A) In some embodiments, the adenosine deaminase comprises amutation at position 613 of the amino acid sequence, which has anasparagine residue in the wild type sequence. In some embodiments, theasparagine residue at position 613 is replaced by a glutamic acidresidue (N613E).

In some embodiments, to improve editing efficiency, the adenosinedeaminase may comprise one or more of the mutations: G336D, G487A,G487V, E488Q, E488H, E488R, E488N, E488A, E488S, E488M, T490C, T490S,V493T, V493S, V493A, V493R, V493D, V493P, V493G, N597K, N597R, N597A,N597E, N597H, N597G, N597Y, A589V, S599T, N613K, N613R, N613A, N613E,based on amino acid sequence positions of hADAR2-D, and mutations in ahomologous ADAR protein corresponding to the above.

In some embodiments, to reduce editing efficiency, the adenosinedeaminase may comprise one or more of the mutations: E488F, E488L,E488W, T490A, T490F, T490Y, T490R, T490K, T490P, T490E, N597F, based onamino acid sequence positions of hADAR2-D, and mutations in a homologousADAR protein corresponding to the above. In particular embodiments, itcan be of interest to use an adenosine deaminase enzyme with reducedefficicay to reduce off-target effects.

In some embodiments, to reduce off-target effects, the adenosinedeaminase comprises one or more of mutations at R348, V351, T375, K376,E396, C451, R455, N473, R474, K475, R477, R481, S486, E488, T490, S495,R510, based on amino acid sequence positions of hADAR2-D, and mutationsin a homologous ADAR protein corresponding to the above. In someembodiments, the adenosine deaminase comprises mutation at E488 and oneor more additional positions selected from R348, V351, T375, K376, E396,C451, R455, N473, R474, K475, R477, R481, S486, T490, S495, R510. Insome embodiments, the adenosine deaminase comprises mutation at T375,and optionally at one or more additional positions. In some embodiments,the adenosine deaminase comprises mutation at N473, and optionally atone or more additional positions. In some embodiments, the adenosinedeaminase comprises mutation at V351, and optionally at one or moreadditional positions. In some embodiments, the adenosine deaminasecomprises mutation at E488 and T375, and optionally at one or moreadditional positions. In some embodiments, the adenosine deaminasecomprises mutation at E488 and N473, and optionally at one or moreadditional positions. In some embodiments, the adenosine deaminasecomprises mutation E488 and V351, and optionally at one or moreadditional positions. In some embodiments, the adenosine deaminasecomprises mutation at E488 and one or more of T375, N473, and V351.

In some embodiments, to reduce off-target effects, the adenosinedeaminase comprises one or more of mutations selected from R348E, V351L,T375G, T375S, R455G, R455S, R455E, N473D, R474E, K475Q, R477E, R481E,S486T, E488Q, T490A, T490S, S495T, and R510E, based on amino acidsequence positions of hADAR2-D, and mutations in a homologous ADARprotein corresponding to the above. In some embodiments, the adenosinedeaminase comprises mutation E488Q and one or more additional mutationsselected from R348E, V351L, T375G, T375S, R455G, R455S, R455E, N473D,R474E, K475Q, R477E, R481E, S486T, T490A, T490S, S495T, and R510E. Insome embodiments, the adenosine deaminase comprises mutation T375G orT375S, and optionally one or more additional mutations. In someembodiments, the adenosine deaminase comprises mutation N473D, andoptionally one or more additional mutations. In some embodiments, theadenosine deaminase comprises mutation V351L, and optionally one or moreadditional mutations. In some embodiments, the adenosine deaminasecomprises mutation E488Q, and T375G or T375G, and optionally one or moreadditional mutations. In some embodiments, the adenosine deaminasecomprises mutation E488Q and N473D, and optionally one or moreadditional mutations. In some embodiments, the adenosine deaminasecomprises mutation E488Q and V351L, and optionally one or moreadditional mutations. In some embodiments, the adenosine deaminasecomprises mutation E488Q and one or more of T375G/S, N473D and V351L.

In certain embodiments, improvement of editing and reduction ofoff-target modification is achieved by chemical modification of gRNAs.gRNAs which are chemically modified as exemplified in Vogel et al.(2014), Angew Chem Int Ed, 53:6267-6271, doi:10.1002/anie.201402634(incorporated herein by reference in its entirety) reduce off-targetactivity and improve on-target efficiency. 2′-O-methyl andphosphothioate modified guide RNAs in general improve editing efficiencyin cells.

ADAR has been known to demonstrate a preference for neighboringnucleotides on either side of the edited A(www.nature.com/nsmb/journal/v23/n5/full/nsmb.3203.html, Matthews et al.(2017), Nature Structural Mol Biol, 23(5): 426-433, incorporated hereinby reference in its entirety). Accordingly, in certain embodiments, thegRNA, target, and/or ADAR is selected optimized for motif preference.

Intentional mismatches have been demonstrated in vitro to allow forediting of non-preferred motifs(academic.oup.com/nar/article-lookup/doi/10.1093/nar/gku272; Schneideret al (2014), Nucleic Acid Res, 42(10):e87); Fukuda et al. (2017),Scientific Reports, 7, doi:10.1038/srep41478, incorporated herein byreference in its entirety). Accordingly, in certain embodiments, toenhance RNA editing efficiency on non-preferred 5′ or 3′ neighboringbases, intentional mismatches in neighboring bases are introduced.

Results suggest that As opposite Cs in the targeting window of the ADARdeaminase domain are preferentially edited over other bases.Additionally, As base-paired with Us within a few bases of the targetedbase show low levels of editing by Cas13b-ADAR fusions, suggesting thatthere is flexibility for the enzyme to edit multiple A's. See e.g. FIG.18. These two observations suggest that multiple As in the activitywindow of Cas13b-ADAR fusions could be specified for editing bymismatching all As to be edited with Cs. Accordingly, in certainembodiments, multiple A:C mismatches in the activity window are designedto create multiple A:I edits. In certain embodiments, to suppresspotential off-target editing in the activity window, non-target As arepaired with As or Gs.

The terms “editing specificity” and “editing preference” are usedinterchangeably herein to refer to the extent of A-to-I editing at aparticular adenosine site in a double-stranded substrate. In someembodiment, the substrate editing preference is determined by the 5′nearest neighbor and/or the 3′ nearest neighbor of the target adenosineresidue. In some embodiments, the adenosine deaminase has preference forthe 5′ nearest neighbor of the substrate ranked as U>A>C>G (“>”indicates greater preference). In some embodiments, the adenosinedeaminase has preference for the 3′ nearest neighbor of the substrateranked as G>C˜A>U (“>” indicates greater preference; “˜” indicatessimilar preference). In some embodiments, the adenosine deaminase haspreference for the 3′ nearest neighbor of the substrate ranked asG>C>U˜A (“>” indicates greater preference; “˜” indicates similarpreference). In some embodiments, the adenosine deaminase has preferencefor the 3′ nearest neighbor of the substrate ranked as G>C>A>U (“>”indicates greater preference). In some embodiments, the adenosinedeaminase has preference for the 3′ nearest neighbor of the substrateranked as C˜G˜A>U (“>” indicates greater preference; “˜” indicatessimilar preference). In some embodiments, the adenosine deaminase haspreference for a triplet sequence containing the target adenosineresidue ranked as TAG>AAG>CAC>AAT>GAA>GAC (“>” indicates greaterpreference), the center A being the target adenosine residue.

In some embodiments, the substrate editing preference of an adenosinedeaminase is affected by the presence or absence of a nucleic acidbinding domain in the adenosine deaminase protein. In some embodiments,to modify substrate editing preference, the deaminase domain isconnected with a double-strand RNA binding domain (dsRBD) or adouble-strand RNA binding motif (dsRBM). In some embodiments, the dsRBDor dsRBM may be derived from an ADAR protein, such as hADAR1 or hADAR2.In some embodiments, a full length ADAR protein that comprises at leastone dsRBD and a deaminase domain is used. In some embodiments, the oneor more dsRBM or dsRBD is at the N-terminus of the deaminase domain. Inother embodiments, the one or more dsRBM or dsRBD is at the C-terminusof the deaminase domain.

In some embodiments, the substrate editing preference of an adenosinedeaminase is affected by amino acid residues near or in the activecenter of the enzyme. In some embodiments, to modify substrate editingpreference, the adenosine deaminase may comprise one or more of themutations: G336D, G487R, G487K, G487W, G487Y, E488Q, E488N, T490A,V493A, V493T, V493S, N597K, N597R, A589V, S599T, N613K, N613R, based onamino acid sequence positions of hADAR2-D, and mutations in a homologousADAR protein corresponding to the above.

Particularly, in some embodiments, to reduce editing specificity, theadenosine deaminase can comprise one or more of mutations E488Q, V493A,N597K, N613K, based on amino acid sequence positions of hADAR2-D, andmutations in a homologous ADAR protein corresponding to the above. Insome embodiments, to increase editing specificity, the adenosinedeaminase can comprise mutation T490A.

In some embodiments, to increase editing preference for target adenosine(A) with an immediate 5′ G, such as substrates comprising the tripletsequence GAC, the center A being the target adenosine residue, theadenosine deaminase can comprise one or more of mutations G336D, E488Q,E488N, V493T, V493S, V493A, A589V, N597K, N597R, S599T, N613K, N613R,based on amino acid sequence positions of hADAR2-D, and mutations in ahomologous ADAR protein corresponding to the above.

Particularly, in some embodiments, the adenosine deaminase comprisesmutation E488Q or a corresponding mutation in a homologous ADAR proteinfor editing substrates comprising the following triplet sequences: GAC,GAA, GAU, GAG, CAU, AAU, UAC, the center A being the target adenosineresidue.

In some embodiments, the adenosine deaminase comprises the wild-typeamino acid sequence of hADAR1-D. In some embodiments, the adenosinedeaminase comprises one or more mutations in the hADAR1-D sequence, suchthat the editing efficiency, and/or substrate editing preference ofhADAR1-D is changed according to specific needs.

In some embodiments, the adenosine deaminase comprises a mutation atGlycine1007 of the hADAR1-D amino acid sequence, or a correspondingposition in a homologous ADAR protein. In some embodiments, the glycineresidue at position 1007 is replaced by a non-polar amino acid residuewith relatively small side chains. For example, in some embodiments, theglycine residue at position 1007 is replaced by an alanine residue(G1007A). In some embodiments, the glycine residue at position 1007 isreplaced by a valine residue (G1007V). In some embodiments, the glycineresidue at position 1007 is replaced by an amino acid residue withrelatively large side chains. In some embodiments, the glycine residueat position 1007 is replaced by an arginine residue (G1007R). In someembodiments, the glycine residue at position 1007 is replaced by alysine residue (G1007K). In some embodiments, the glycine residue atposition 1007 is replaced by a tryptophan residue (G1007W). In someembodiments, the glycine residue at position 1007 is replaced by atyrosine residue (G1007Y). Additionally, in other embodiments, theglycine residue at position 1007 is replaced by a leucine residue(G1007L). In other embodiments, the glycine residue at position 1007 isreplaced by a threonine residue (G1007T). In other embodiments, theglycine residue at position 1007 is replaced by a serine residue(G1007S).

In some embodiments, the adenosine deaminase comprises a mutation atglutamic acid1008 of the hADAR1-D amino acid sequence, or acorresponding position in a homologous ADAR protein. In someembodiments, the glutamic acid residue at position 1008 is replaced by apolar amino acid residue having a relatively large side chain. In someembodiments, the glutamic acid residue at position 1008 is replaced by aglutamine residue (E1008Q). In some embodiments, the glutamic acidresidue at position 1008 is replaced by a histidine residue (E1008H). Insome embodiments, the glutamic acid residue at position 1008 is replacedby an arginine residue (E1008R). In some embodiments, the glutamic acidresidue at position 1008 is replaced by a lysine residue (E1008K). Insome embodiments, the glutamic acid residue at position 1008 is replacedby a nonpolar or small polar amino acid residue. In some embodiments,the glutamic acid residue at position 1008 is replaced by aphenylalanine residue (E1008F). In some embodiments, the glutamic acidresidue at position 1008 is replaced by a tryptophan residue (E1008W).In some embodiments, the glutamic acid residue at position 1008 isreplaced by a glycine residue (E1008G). In some embodiments, theglutamic acid residue at position 1008 is replaced by an isoleucineresidue (E1008I). In some embodiments, the glutamic acid residue atposition 1008 is replaced by a valine residue (E1008V). In someembodiments, the glutamic acid residue at position 1008 is replaced by aproline residue (E1008P). In some embodiments, the glutamic acid residueat position 1008 is replaced by a serine residue (E1008S). In otherembodiments, the glutamic acid residue at position 1008 is replaced byan asparagine residue (E1008N). In other embodiments, the glutamic acidresidue at position 1008 is replaced by an alanine residue (E1008A). Inother embodiments, the glutamic acid residue at position 1008 isreplaced by a Methionine residue (E1008M). In some embodiments, theglutamic acid residue at position 1008 is replaced by a leucine residue(E1008L).

In some embodiments, to improve editing efficiency, the adenosinedeaminase may comprise one or more of the mutations: E1007S, E1007A,E1007V, E1008Q, E1008R, E1008H, E1008M, E1008N, E1008K, based on aminoacid sequence positions of hADAR1-D, and mutations in a homologous ADARprotein corresponding to the above.

In some embodiments, to reduce editing efficiency, the adenosinedeaminase may comprise one or more of the mutations: E1007R, E1007K,E1007Y, E1007L, E1007T, E1008G, E1008I, E1008P, E1008V, E1008F, E1008W,E1008S, E1008N, E1008K, based on amino acid sequence positions ofhADAR1-D, and mutations in a homologous ADAR protein corresponding tothe above.

In some embodiments, the substrate editing preference, efficiency and/orselectivity of an adenosine deaminase is affected by amino acid residuesnear or in the active center of the enzyme. In some embodiments, theadenosine deaminase comprises a mutation at the glutamic acid 1008position in hADAR1-D sequence, or a corresponding position in ahomologous ADAR protein. In some embodiments, the mutation is E1008R, ora corresponding mutation in a homologous ADAR protein. In someembodiments, the E1008R mutant has an increased editing efficiency fortarget adenosine residue that has a mismatched G residue on the oppositestrand.

In some embodiments, the adenosine deaminase protein further comprisesor is connected to one or more double-stranded RNA (dsRNA) bindingmotifs (dsRBMs) or domains (dsRBDs) for recognizing and binding todouble-stranded nucleic acid substrates. In some embodiments, theinteraction between the adenosine deaminase and the double-strandedsubstrate is mediated by one or more additional protein factor(s),including a CRISPR/CAS protein factor. In some embodiments, theinteraction between the adenosine deaminase and the double-strandedsubstrate is further mediated by one or more nucleic acid component(s),including a guide RNA.

In certain example embodiments, directed evolution may be used to designmodified ADAR proteins capable of catalyzing additional reactionsbesides deamination of a adenine to a hypoxanthine. For example

According to the present invention, the substrate of the adenosinedeaminase is an RNA/DNAn RNA duplex formed upon binding of the guidemolecule to its DNA target which then forms the CRISPR-Cas complex withthe CRISPR-Cas enzyme. The RNA/DNA or DNA/RNAn RNA duplex is alsoreferred to herein as the “RNA/DNA hybrid”, “DNA/RNA hybrid” or“double-stranded substrate”. The particular features of the guidemolecule and CRISPR-Cas enzyme are detailed below.

The term “editing selectivity” as used herein refers to the fraction ofall sites on a double-stranded substrate that is edited by an adenosinedeaminase. Without being bound by theory, it is contemplated thatediting selectivity of an adenosine deaminase is affected by thedouble-stranded substrate's length and secondary structures, such as thepresence of mismatched bases, bulges and/or internal loops.

In some embodiments, when the substrate is a perfectly base-pairedduplex longer than 50 bp, the adenosine deaminase may be able todeaminate multiple adenosine residues within the duplex (e.g., 50% ofall adenosine residues). In some embodiments, when the substrate isshorter than 50 bp, the editing selectivity of an adenosine deaminase isaffected by the presence of a mismatch at the target adenosine site.Particularly, in some embodiments, adenosine (A) residue having amismatched cytidine (C) residue on the opposite strand is deaminatedwith high efficiency. In some embodiments, adenosine (A) residue havinga mismatched guanosine (G) residue on the opposite strand is skippedwithout editing.

RNA Editing with ADAR1/ADAR2

Inherited Diseases with G to A Changes (in Transcribed/CodingRegions/Splicing Sequences) may be Treated by the Systems and MethodsDisclosed Herein.

ADAR mutates adenosine (A) to insoine (I) which is treated by thecellular translational machinery as guanosine (G). In some embodiments,RNA editing with dCas13-ADAR fusions may be used to reverse the effectsof genetic mutations in transcripts containing pathogenic G to Achanges. ADAR RNA editing may occur in double-stranded RNA, where theedited base is opposite a U or a C. Cas13b-ADAR (e.g., dCas13b-ADAR) RNAediting may be achieved by using the guide-RNA to create adouble-stranded RNA structure and fusing ADAR to a dead version ofCas13b genetically.

Out of all pre-termination pathogenic mutations in ClinVar, 985 out of9135 (10.7%) are G to A mutations and potential candidates forcorrection by Cas13-ADAR fusion editors. Out of all pathogenic mutationsin ClinVar, 15531 out of 97941 (15.8%) are G to A mutations.

Examples of diseases with G>A mutations that can treated by the systemsand methods herein include: Meier-Gorlin syndrome, Seckel syndrome 4,Joubert syndrome 5, Leber congenital amaurosis 10, Charcot-Marie-Toothdisease, type 2, Leukoencephalopathy, Usher syndrome, type 2C,Spinocerebellar ataxia 28, Glycogen storage disease type III, Primaryhyperoxaluria, type I, Long QT syndrome 2, Sjögren-Larsson syndrome,Hereditary fructosuria, Neuroblastoma, Amyotrophic lateral sclerosistype 9, Kallmann syndrome 1, Limb-girdle muscular dystrophy, type 2L,Familial adenomatous polyposis 1, Familial type 3 hyperlipoproteinemia,Alzheimer disease, type 1, Metachromatic leukodystrophy.

The following diseases may also be treated using the method and systemsherein.

Premature Termination Diseases

Pre-termination diseases are characterized by mutations in early stopcodons, either through single nucleotide polymorphisms that introducetermination, indels that change the translational frame of the proteinand generate new stop codons, or alternative splicing thatpreferentially introduces exons that have early termination. By removingstop codons generated in these ways via A to I editing, RNA editing withthe systems and methods herein with ADAR may rescue diseases involvingpremature termination. In cases where SNPs are not G to A, but generatenonsense mutations, some clinical benefit may be derived from changingnonsense mutations into missense mutations.

Change Fertility Mutations without Germline Editing

One advantage of RNA editing over DNA editing is in cases of SNPsaffecting fertility, where correction with genome editing wouldnecessarily result in germline editing, with potential ethical or safetyimplications. RNA editing with the systems and methods herein maycorrect these mutations without permanent effects on the genome, therebycircumventing these issues.

Splicing Alteration

Pre-mRNA requires specific splice donor and acceptor sequences in orderto undergo processing by the spliceosome. Splice acceptor sites containan invariant AG sequence that is necessary for acceptance of the attackby the splice donor sequence and intron removal. By targetingCas13b-ADAR fusions to pre-mRNA and editing AG splice acceptor sites toIG, the splice acceptor site may be inactivated, resulting in skippingof the downstream exon. This approach to splicing alteration hasadvantages over the current method of exon skipping with chemicallymodified anti-sense oligos. Cas13b-ADAR may be genetically encoded,allowing for long-term exon skipping. Additionally, Cas13b-ADAR maycreate a mutation to promote skipping, which is likely to be more robustthan masking of the splice donor/acceptor site by a double stranded RNA,as is done with anti-sense oligos.

Neoantigens

Neoantigens in cancer are novel antigens that are expressed in tumorcells due to mutations that arise because of defective mismatch repair.Engineering T cells against neoantigens is advantageous because the Tcells will have no off-target activity and thus toxicity since theantigens are only expressed in the tumor cells. With RNA editing withthe systems and methods herein, the Cas13-ADAR fusions may be targetedto cancer cells to introduce mutations in transcripts that may introduceamino acid changes and new antigens that could be targeted usingchimeric antigen receptor T cells. This approach is better than DNA baseeditors because it is transient and thus the risk of editing non-tumorcells permanently due to off-target delivery is minimal.

Changing microRNA Targets (for Tumor Suppressors)

ADAR naturally edits mRNA to generate or remove microRNA targets,thereby modulating expression. Programmable RNA editing can be used toup- or down-regulate microRNA targets via altering of targeting regions.Additionally, microRNAs themselves are natural substrates for ADAR andprogrammable RNA editing of micoRNAs can reduce or enhance the functionon their corresponding targets.

Make Multiple Edits along a Region (Multiple Mismatches in Guide)

The Cas13-ADAR fusions can be precisely targeted to edit specificadenosines by introducing a mismatch in the guide region across from thedesired adenosine target and creating a bubble that is favorable forA-to-I editing. By introducing multiple of these mismatches acrossdifferent adenosine sites in the guide/target duplex, multiple mutationsmay be introduced at once.

Reversal of TAA (Double A to G) for PTC

Many diseases that involve pretermination codon changes involve a TAAstop codon, which would require to A-to-I changes to correct rather thanthe TAG or TGA stop codons which only need one A-to-I edit. Twoapproaches can be used to reverse the TAA stop codon. (1) As describedherein, two mismatches may be introduced in the guide against the twoadenosines in the TAA codon. (2) A two guide array may be used toconvert each of the adenosines to inosine sequentially. The first guidein the array may contain a mutation against the first adenosine and thesecond guide may then have complementarity to this change and have amismatch against the second adenosine in the stop codon.

Cancer (GOF, LOF Mutation Reversal)

Many oncogenic changes in cancer involve G to A mutations that introducegain of function or loss of function phenotypes to the mutated proteins.The RNA editing systems and methods herein may be well positioned tocorrect these changes and reduce oncogenesis.

Design of New Base Preferences

Current ADAR1/2 proteins have been found to have surrounding basepreferences for catalytic activity (,pubs.acs.org/doi/10.102/acschembio.5b00711), which may pose constraintsfor certain applications. Rational mutagenesis or directed evolution ofADAR variants with altered or relaxed base preferences can increase theversatility of programmable RNA editing.

ADAR Orthologs that can be Used

Various ADAR orthologs may be used for the methods and systems herein.Examples of ADAR orthologs that may be used include:

TABLE 1 ADAR Name Protein Sequence HomoQLHLPQVLADAVSRLVLGKFGDLTDNFSSPHARRKVLAGVVMTTGTDVKDAsapiens_ADAR2_E_Q_MutantKVISVSTGTKCINGEYMSDRGLALNDCHAEIISRRSLLRFLYTQLELYLNNKDDQKRSIFQKSERGGFRLKENVQFHLYISTSPCGDARIFSPHEPILEEPADRHPNRKARGQLRTKIESGQGTIPVRSNASIQTWDGVLQGERLLTMSCSDKIARWNVVGIQGSLLSIFVEPIYFSSIILGSLYHGDHLSRAMYQRISNIEDLPPLYTLNKPLLSGISNAEARQPGKAPNFSVNWTVGDSAIEVINATTGKDELGRASRLCKHALYCRWMRVHGKVPSHLLRSKITKPNVYHESKLAAKEYQAAKARLFTAFIKAGLGAWVE KPTEQDQFSLT* HomoSLGTGNRCVKGDSLSLKGETVNDCHAEIISRRGFIRFLYSELMKYNSQTAKDSIsapiens_ADAR1_E_Q_MutantFEPAKGGEKLQIKKTVSFHLYISTAPCGDGALFDKSCSDRAMESTESRHYPVFENPKQGKLRTKVENGQGTIPVESSDIVPTWDGIRLGERLRTMSCSDKILRWNVLGLQGALLTHFLQPIYLKSVTLGYLFSQGHLTRAICCRVTRDGSAFEDGLRHPFIVNHPKVGRVSIYDSKRQSGKTKETSVNWCLADGYDLEILDGTRGTVDGPRNELSRVSKKNIFLLFKKLCSFRYRRDLLRLSYGEAKKAARDYETAKNYFKKGLKD MGYGNWISKPQEEKNFOctopus SVGTGNRCLTGDHLSLEGNSVNDSHAEMITRRGFLRYLYRHLLEYDAEVPNDLvulgaris_ADAR1_E_Q_MutantFEKGERSICRIKTNITFHLYISTAPCGDGALFSPRDTDSSNAKMEEENKHIHNPTFSSSVQGLLRTKVEGGQGTIPIDADFTEQTWDGIQRGERLRTMSCSDKICRWNVVGLQGALLSHFIEPIYLDSLTLGYLYDHGHLARAVCCRIERGEASVNQLLPEGYRLNHPWLGRVTACDPPRETQKTKSLSINWCYDDEKSEVLDGTAGICYTAIEKNLFSRLTKHNLYEEFKRVCRKFDRNDLLTAPSYNKAKMMATPFQTAKNVMLK KLKENNCGTWVSKPIEEEMFSepia_ADAR1_E_Q_MutantSVGTGNRCLTGDRLSLEGNSVNDSHAEMVTRRGFLRYLYKHLLEYDPEKPHDLFEKGERSLCRIKTNITFHLYISTAPCGDGALFSPRDTDSSNVKVDEENKHVHNPTFSSSVQGLLRTKVEGGQGTIPIDADFTEQTWDGIQRGERLRTMSCSDKICRWNVVGLQGALLSHFVEPIYLESLTLGYLYDHGHLARAVCCRIERGEASVNQLLPEGYRLNHPWLGRVTACDPPRETQKTKSLSINWCYDDEKSEVLDGTAGICYTAIEKNLFSRLTKHSLYEEFKKVCQKFEREDLLNVTSYNKAKMMAIPFQTAKNVMLKKLKENNCGTWVSKPIEEEMF OctopusGIGTGTKCINGEHMSDRGFGVNDCHAEIIARRCFLRYIYDQLELHLSDNSDVRNvulgaris_ADAR2_E_Q_MutantSSIFELRDKGGYQLKENIQFHLYISTAPCGDARIFSPHGQDVETGDRHPNRKARGQLRTKIESGQGTIPVRTSGVIQTWDGVLEGERLLTMSCSDKIARWNVLGIQGSLLSHFMNPIYLESIILGSLYHSDHLSRAMYSRISIIENLPEPFHLNRPFLSGISSPESRQPGKAPNFGINWRKEDETFEVINAMTGRVEGGSVSRICKQALFGRFMSLYGKLSSLTGQSVTTRPTHYSDAKAAVMEYQLAKQCVFQAFQKAGLGNWVQKPIEQ DQFSepia_ADAR2_E_Q_MutantGIGTGTKCINGEYMNDRGFAVNDCHAEIIARRCFLRFIYDQLEMHLSEDPEVRGQSVFELRDGGGYKLKPNIHFHLYISTAPCGDARIFSPHGQDVETGDRHPNRKARGQLRTKIESGQGTIPVRSSGFIQTWDGVLEGERLLTMSCSDKIARWNVLGIQGALLCHFMHPIYLESIILGSLYHSDHLSRAVYCRIASIENLPDLFQLNRPFLSGISSPESRQPGKAPNFGINWRRNDDTFEVINAMTGRVEGGNMSRICKQALFDRFMNLYGRLSSLTGQSVTTRPTLYSEAKAAVMEYQLAKQCVFQAFQKAGLGNWVQKPI EQDQF DoryteusthisGIGTGTKCINGEYMNDRGFAVNDCHAEIIARRCFLRFIYDQLELHLSDNAEVRGopalescens_ADAR2_E_Q_MutantQSIFELRDAGGYKLKPNIQFHLYISTAPCGDARIFSPHGQDVETGDRHPNRKARGQLRTKIESGQGTIPVRSSGFIQTWDGVLEGERLLTMSCSDKIARWNVLGVQGALLCHFMHPIYLESIILGSLYHSDHLSRAVYCRIAAIENLPDLFRLNRPFLSGISSPESRQPGKAPNFGINWRRNDDSFEVINAMTGRVEGGSMSRICKQALFDRFMNLYGKLSSLTGQSVTTRPALYSEAKATVMEYQLAKQCVFQAFQKAGLGNWVQK PIEQDQF

Note that the orthologs above are listed with the hyperactive E to Qmutation. The cephalopod orthologs may be used. Cephalopods are knownfor extremely high rates of RNA editing and so these orthologs may havehigher activity than the human variants or altered base preferences.

An alignment tree of these orthologs is shown in FIG. 74

ADAR mutants with increased activity in human cells. ADAR mutants withaltered activity in vitro or in yeast have been previously reported. Thepresent disclosure further provides for screening or rational design ofmutants with increased activity in the context of human cells, which mayimprove the efficiency or specificity of ADAR-based programmable RNAediting systems or constructs

Biological applications of inosine generation. RNA editing with ADARgenerates inosine, which, when occurring multiple times in a transcript,can interact with endogenous biological pathways to increaseinflammation in cells and tissues. Generation of multiple inosine basescould increase inflammation, especially in cells where inflammation canlead to clearance. Additional inosine generation may also be used todestabilize transcripts.

Removing upstream start codons to promote protein expression ofdownstream ORF (ATG mutation). Anti-sense oligos may be used forblocking upstream start codon sites to promote protein expression atdownstream start codons. This may allow the boosting of endogenousprotein levels for therapeutic purposes. Cas13-ADAR fusions mayaccomplish a similar effect by convert ATG sites to ITG (GTG) sites andthus remove upstream codons in endogenous transcripts and thus boostprotein translation. This may be an application that allows for boostinggene expression. An example includes boosting fetal hemoglobin levels insickle cell disease and thalassemias.

Mutagenesis of ADAR for C to U or any transition. The ADARs (e.g., thoselisted in the orthologs section) may be made into C to U editors oreditors of any base transition through rational mutagenesis or directedevolution.

APOBEC RNA Editing

C to U conversions. The systems may comprise Cas13b-APOBEC fusions(e.g., dCas13b-APOBEC fusions). These fusions may allow C-to-U editingof RNA. APOBEC substrates are ssRNA and these systems may target regionsof the RNA around the guide/target duplex.

Knockdown with C to U via stop codon introduction. In addition tocorrecting pathogenic U to C mutations that arise during the cellularlife cycle, Cas13-APOBEC fusions may allow for introducing stop codonsby converting a CAA, CGA, or CAG to TAA, TGA, or TAG, respectively.

APOBEC Orthologs. APOBEC orthologs may be screened using the systems.Screening additional APOBEC orthologs in fusion with Cas13 (e.g.,dCas13) may increase the efficiency of C-to-U editing, or may allow foradditional types of base conversions.

Mutating APOBEC for dsRNA/base flip/increased activity. RNAinsertions/deletions with editosome. The systems may further compriseRNA editosomes (e.g., RNA editosome fused to Cas13 such as dCas13). Suchsystems may guide targeted deletion or insertion of sequences intotranscripts

References

Human SNPs resulting in premature stop codons and protein truncation.Hum Genomics. 2006; 2(5): 274-286.

The genetics of human infertility by functional interrogation of SNPs inmice. PNAS Aug. 18, 2015 112 (33) 10431-10436

microRNA editing in seed region aligns with cellular changes in hypoxicconditions. Nucleic Acids Research, Volume 44, Issue 13, 27 Jul. 2016,Pages 6298-6308

RNA Editing Modulates Human Hepatic Aryl Hydrocarbon Receptor Expressionby Creating MicroRNA Recognition Sequence. Jan. 8, 2016, The Journal ofBiological Chemistry, 291, 894-903.

Mechanistic insights into editing-site specificity of ADARs. PNAS Nov.27, 2012 109 (48) E3295-E3304.

A Phenotypic Screen for Functional Mutants of Human Adenosine DeaminaseActing on RNA 1. ACS Chem. Biol., 2015, 10 (11), pp 2512-2519.

Inosine-Containing RNA Is a Novel Innate Immune Recognition Element andReduces RSV Infection. PLoS One. 2011; 6(10):e26463.

Inosine-Mediated Modulation of RNA Sensing by Toll-Like Receptor 7(TLR7) and TLR8. J Virol. 2014 January; 88(2):799-810.

Specific cleavage of hyper-edited dsRNAs. The EMBO Journal (2001) 20,4243-4252.

Translation efficiency of mRNAs is increased by antisenseoligonucleotides targeting upstream open reading frames. NatureBiotechnology volume 34, pages 875-880 (2016).

The mechanism of U insertion/deletion RNA editing in kinetoplastidmitochondria. Nucleic Acids Res. 1997 Oct. 1; 25(19): 3751-3759.

Functions and Regulation of RNA Editing by ADAR Deaminases. AnnualReview of Biochemistry, Vol. 79:321-349 (Volume publication date Jul. 7,2010).

Trade-off between Transcriptome Plasticity and Genome Evolution inCephalopods. Cell. Volume 169, Issue 2, 6 Apr. 2017, Pages 191-202.e11.

An adenosine-to-inosine tRNA-editing enzyme that can perform C-to-Udeamination of DNA. PNAS May 8, 2007 104 (19) 7821-7826.

Cytidine deaminase

The term “cytidine deaminase” or “cytidine deaminase protein” as usedherein refers to a protein, a polypeptide, or one or more functionaldomain(s) of a protein or a polypeptide that is capable of catalyzing ahydrolytic deamination reaction that converts an cytosine (or ancytosine moiety of a molecule) to an uracil (or a uracil moiety of amolecule), as shown below. In some embodiments, the cytosine-containingmolecule is an cytidine (C), and the uracil-containing molecule is anuridine (U). The cytosine-containing molecule can be deoxyribonucleicacid (DNA) or ribonucleic acid (RNA).

According to the present disclosure, cytidine deaminases that can beused in connection with the present disclosure include, but are notlimited to, members of the enzyme family known as apolipoprotein BmRNA-editing complex (APOBEC) family deaminase, an activation-induceddeaminase (AID), or a cytidine deaminase 1 (CDA1). In particularembodiments, the deaminase in an APOBEC1 deaminase, an APOBEC2deaminase, an APOBEC3A deaminase, an APOBEC3B deaminase, an APOBEC3Cdeaminase, and APOBEC3D deaminase, an APOBEC3E deaminase, an APOBEC3Fdeaminase an APOBEC3G deaminase, an APOBEC3H deaminase, or an APOBEC4deaminase.

In the methods and systems of the present invention, the cytidinedeaminase is capable of targeting Cytosine in a DNA single strand. Incertain example embodiments the cytidine deaminase may edit on a singlestrand present outside of the binding component e.g. bound Cas13. Inother example embodiments, the cytidine deaminase may edit at alocalized bubble, such as a localized bubble formed by a mismatch at thetarget edit site but the guide sequence. In certain example embodimentsthe cytidine deaminase may contain mutations that help focus the are ofactivity such as those disclosed in Kim et al., Nature Biotechnology(2017) 35(4):371-377 (doi:10.1038/nbt.3803.

In some embodiments, the cytidine deaminase is derived from one or moremetazoa species, including but not limited to, mammals, birds, frogs,squids, fish, flies and worms. In some embodiments, the cytidinedeaminase is a human, primate, cow, dog rat or mouse cytidine deaminase.

In some embodiments, the cytidine deaminase is a human APOBEC, includinghAPOBEC1 or hAPOBEC3. In some embodiments, the cytidine deaminase is ahuman AID.

In some embodiments, the cytidine deaminase protein recognizes andconverts one or more target cytosine residue(s) in a single-strandedbubble of a RNA duplex into uracil residues (s). In some embodiments,the cytidine deaminase protein recognizes a binding window on thesingle-stranded bubble of a RNA duplex. In some embodiments, the bindingwindow contains at least one target cytosine residue(s). In someembodiments, the binding window is in the range of about 3 bp to about100 bp. In some embodiments, the binding window is in the range of about5 bp to about 50 bp. In some embodiments, the binding window is in therange of about 10 bp to about 30 bp. In some embodiments, the bindingwindow is about 1 bp, 2 bp, 3 bp, 5 bp, 7 bp, 10 bp, 15 bp, 20 bp, 25bp, 30 bp, 40 bp, 45 bp, 50 bp, 55 bp, 60 bp, 65 bp, 70 bp, 75 bp, 80bp, 85 bp, 90 bp, 95 bp, or 100 bp.

In some embodiments, the cytidine deaminase protein comprises one ormore deaminase domains. Not intended to be bound by theory, it iscontemplated that the deaminase domain functions to recognize andconvert one or more target cytosine (C) residue(s) contained in asingle-stranded bubble of a RNA duplex into (an) uracil (U) residue (s).In some embodiments, the deaminase domain comprises an active center. Insome embodiments, the active center comprises a zinc ion. In someembodiments, amino acid residues in or near the active center interactwith one or more nucleotide(s) 5′ to a target cytosine residue. In someembodiments, amino acid residues in or near the active center interactwith one or more nucleotide(s) 3′ to a target cytosine residue.

In some embodiments, the cytidine deaminase comprises human APOBEC1 fullprotein (hAPOBEC1) or the deaminase domain thereof (hAPOBEC1-D) or aC-terminally truncated version thereof (hAPOBEC-T). In some embodiments,the cytidine deaminase is an APOBEC family member that is homologous tohAPOBEC1, hAPOBEC-D or hAPOBEC-T. In some embodiments, the cytidinedeaminase comprises human AID1 full protein (hAID) or the deaminasedomain thereof (hAID-D) or a C-terminally truncated version thereof(hAID-T). In some embodiments, the cytidine deaminase is an AID familymember that is homologous to hAID, hAID-D or hAID-T. In someembodiments, the hAID-T is a hAID which is C-terminally truncated byabout 20 amino acids.

In some embodiments, the cytidine deaminase comprises the wild-typeamino acid sequence of a cytosine deaminase. In some embodiments, thecytidine deaminase comprises one or more mutations in the cytosinedeaminase sequence, such that the editing efficiency, and/or substrateediting preference of the cytosine deaminase is changed according tospecific needs.

Certain mutations of APOBEC1 and APOBEC3 proteins have been described inKim et al., Nature Biotechnology (2017) 35(4):371-377(doi:10.1038/nbt.3803); and Harris et al. Mol. Cell (2002) 10:1247-1253,each of which is incorporated herein by reference in its entirety.

In some embodiments, the cytidine deaminase is an APOBEC1 deaminasecomprising one or more mutations at amino acid positions correspondingto W90, R118, H121, H122, R126, or R132 in rat APOBEC1, or an APOBEC3Gdeaminase comprising one or more mutations at amino acid positionscorresponding to W285, R313, D316, D317X, R320, or R326 in human APOBEC3G.

In some embodiments, the cytidine deaminase comprises a mutation attryptophane⁹⁰ of the rat APOBEC1 amino acid sequence, or a correspondingposition in a homologous APOBEC protein, such as tryptophane²⁸⁵ ofAPOBEC3G. In some embodiments, the tryptophane residue at position 90 isreplaced by an tyrosine or phenylalanine residue (W90Y or W90F).

In some embodiments, the cytidine deaminase comprises a mutation atArginine¹¹⁸ of the rat APOBEC1 amino acid sequence, or a correspondingposition in a homologous APOBEC protein. In some embodiments, thearginine residue at position 118 is replaced by an alanine residue(R118A).

In some embodiments, the cytidine deaminase comprises a mutation atHistidine¹²¹ of the rat APOBEC1 amino acid sequence, or a correspondingposition in a homologous APOBEC protein. In some embodiments, thehistidine residue at position 121 is replaced by an arginine residue(H121R).

In some embodiments, the cytidine deaminase comprises a mutation atHistidine¹²² of the rat APOBEC1 amino acid sequence, or a correspondingposition in a homologous APOBEC protein. In some embodiments, thehistidine residue at position 122 is replaced by an arginine residue(H122R).

In some embodiments, the cytidine deaminase comprises a mutation atArginine¹²⁶ of the rat APOBEC1 amino acid sequence, or a correspondingposition in a homologous APOBEC protein, such as Arginine³²⁰ ofAPOBEC3G. In some embodiments, the arginine residue at position 126 isreplaced by an alanine residue (R126A) or by a glutamic acid (R126E).

In some embodiments, the cytidine deaminase comprises a mutation atarginine¹³² of the APOBEC1 amino acid sequence, or a correspondingposition in a homologous APOBEC protein. In some embodiments, thearginine residue at position 132 is replaced by a glutamic acid residue(R132E).

In some embodiments, to narrow the width of the editing window, thecytidine deaminase may comprise one or more of the mutations: W90Y,W90F, R126E and R132E, based on amino acid sequence positions of ratAPOBEC1, and mutations in a homologous APOBEC protein corresponding tothe above.

In some embodiments, to reduce editing efficiency, the cytidinedeaminase may comprise one or more of the mutations: W90A, R118A, R132E,based on amino acid sequence positions of rat APOBEC1, and mutations ina homologous APOBEC protein corresponding to the above. In particularembodiments, it can be of interest to use a cytidine deaminase enzymewith reduced efficacy to reduce off-target effects.

In some embodiments, the cytidine deaminase is wild-type rat APOBEC1(rAPOBEC1, or a catalytic domain thereof. In some embodiments, thecytidine deaminase comprises one or more mutations in the rAPOBEC1sequence, such that the editing efficiency, and/or substrate editingpreference of rAPOBEC1 is changed according to specific needs.

rAPOBEC1: MSSETGPVAVDPTLRRRIEPHEFEVFFDPRELRKETCLLYEINWGGRHSIWRHTSQNTNKHVEVNFIEKFTTERYFCPNTRCSITWFLSWSPCGECSRAITEFLSRYPHVTLFIYIARLYHHADPRNRQGLRDLISSGVTIQIMTEQESGYCWRNFVNYSPSNEAHWPRYPHLWVRLYVLELYCIILGLPPCLNILRRKQPQLTFFTIALQSCHYQRLPPHILWATGLK

In some embodiments, the cytidine deaminase is wild-type human APOBEC1(hAPOBEC1) or a catalytic domain thereof. In some embodiments, thecytidine deaminase comprises one or more mutations in the hAPOBEC1sequence, such that the editing efficiency, and/or substrate editingpreference of hAPOBEC1 is changed according to specific needs.

APOBEC1: MTSEKGPSTGDPTLRRRIEPWEFDVFYDPRELRKEACLLYEIKWGMSRKIWRSSGKNTTNHVEVNFIKKFTSERDFHPSMSCSITWFLSWSPCWECSQAIREFLSRHPGVTLVIYVARLFWHMDQQNRQGLRDLVNSGVTIQIMRASEYYHCWRNFVNYPPGDEAHWPQYPPLWMMLYALELHCIILSLPPCLKISRRWQNHLTFFRLHLQNCHYQTIPPHILLATGLIHPSVAWR

In some embodiments, the cytidine deaminase is wild-type human APOBEC3G(hAPOBEC3G) or a catalytic domain thereof. In some embodiments, thecytidine deaminase comprises one or more mutations in the hAPOBEC3Gsequence, such that the editing efficiency, and/or substrate editingpreference of hAPOBEC3G is changed according to specific needs.

hAPOBEC3G: MELKYHPEMRFFHWFSKWRKLHRDQEYEVTWYISWSPCTKCTRDMATFLAEDPKVTLTIFVARLYYFWDPDYQEALRSLCQKRDGPRATMKIMNYDEFQHCWSKFVYSQRELFEPWNNLPKYYILLHIMLGEILRHSMDPPTFTFNFNNEPWVRGRHETYLCYEVERMHNDTWVLLNQRRGFLCNQAPHKHGFLEGRHAELCFLDVIPFWKLDLDQDYRVTCFTSWSPCFSCAQEMAKFISKNKHVSLCIFTARIYDDQGRCQEGLRTLAEAGAKISIMTYSEFKHCWDTFVDHQGCPFQPWDGLDEHSQDLSGRLRAILQNQEN

In some embodiments, the cytidine deaminase is wild-type Petromyzonmarinus CDA1 (pmCDA1) or a catalytic domain thereof. In someembodiments, the cytidine deaminase comprises one or more mutations inthe pmCDA1 sequence, such that the editing efficiency, and/or substrateediting preference of pmCDA1 is changed according to specific needs.

pmCDA1: MTDAEYVRIHEKLDIYTFKKQFFNNKKSVSHRCYVLFELKRRGERRACFWGYAVNKPQSGTERGIHAEIFSIRKVEEYLRDNPGQFTINWYSSWSPCADCAEKILEWYNQELRGNGHTLKIWACKLYYEKNARNQIGLWNLRDNGVGLNVMVSEHYQCCRKIFIQSSHNQLNENRWLEKTLKRAEKRRSELSIMIQVKIL HTTKSPAV

In some embodiments, the cytidine deaminase is wild-type human AID(hAID) or a catalytic domain thereof. In some embodiments, the cytidinedeaminase comprises one or more mutations in the pmCDA1 sequence, suchthat the editing efficiency, and/or substrate editing preference ofpmCDA1 is changed according to specific needs.

hAID: MDSLLMNRRKFLYQFKNVRWAKGRRETYLCYVVKRRDSATSFSLDFGYLRNKNGCHVELLFLRYISDWDLDPGRCYRVTWFTSWSPCYDCARHVADFLRGNPYLSLRIFTARLYFCEDRKAEPEGLRRLHRAGVQIAIMTFKDYFYCWNTFVENHERTFKAWEGLHENSVRLSRQLRRILLPLYEVDDLRDAFRTLGLLD

In some embodiments, the cytidine deaminase is truncated version of hAID(hAID-DC) or a catalytic domain thereof. In some embodiments, thecytidine deaminase comprises one or more mutations in the hAID-DCsequence, such that the editing efficiency, and/or substrate editingpreference of hAID-DC is changed according to specific needs.

hAID-DC: MDSLLMNRRKFLYQFKNVRWAKGRRETYLCYVVKRRDSATSFSLDFGYLRNKNGCHVELLFLRYISDWDLDPGRCYRVTWFTSWSPCYDCARHVADFLRGNPNLSLRIFTARLYFCEDRKAEPEGLRRLHRAGVQIAIMTFKDYFYCWNTFVENHERTFKAWEGLHENSVRLSRQLRRILL

Additional embodiments of the cytidine deaminase are disclosed in WOWO2017/070632, titled “Nucleobase Editor and Uses Thereof,” which isincorporated herein by reference in its entirety.

In some embodiments, the cytidine deaminase has an efficient deaminationwindow that encloses the nucleotides susceptible to deamination editing.Accordingly, in some embodiments, the “editing window width” refers tothe number of nucleotide positions at a given target site for whichediting efficiency of the cytidine deaminase exceeds the half-maximalvalue for that target site. In some embodiments, the cytidine deaminasehas an editing window width in the range of about 1 to about 6nucleotides. In some embodiments, the editing window width of thecytidine deaminase is 1, 2, 3, 4, 5, or 6 nucleotides.

Not intended to be bound by theory, it is contemplated that in someembodiments, the length of the linker sequence affects the editingwindow width. In some embodiments, the editing window width increases(e.g., from about 3 to about 6 nucleotides) as the linker length extends(e.g., from about 3 to about 21 amino acids). In a non-limiting example,a 16-residue linker offers an efficient deamination window of about 5nucleotides. In some embodiments, the length of the guide RNA affectsthe editing window width. In some embodiments, shortening the guide RNAleads to a narrowed efficient deamination window of the cytidinedeaminase.

In some embodiments, mutations to the cytidine deaminase affect theediting window width. In some embodiments, the cytidine deaminasecomponent of the CD-functionalized CRISPR system comprises one or moremutations that reduce the catalytic efficiency of the cytidinedeaminase, such that the deaminase is prevented from deamination ofmultiple cytidines per DNA binding event. In some embodiments,tryptophan at residue 90 (W90) of APOBEC1 or a corresponding tryptophanresidue in a homologous sequence is mutated. In some embodiments, thecatalytically inactive Cas13 is fused to or linked to an APOBEC1 mutantthat comprises a W90Y or W90F mutation. In some embodiments, tryptophanat residue 285 (W285) of APOBEC3G, or a corresponding tryptophan residuein a homologous sequence is mutated. In some embodiments, thecatalytically inactive Cas13 is fused to or linked to an APOBEC3G mutantthat comprises a W285Y or W285F mutation.

In some embodiments, the cytidine deaminase component ofCD-functionalized CRISPR system comprises one or more mutations thatreduce tolerance for non-optimal presentation of a cytidine to thedeaminase active site. In some embodiments, the cytidine deaminasecomprises one or more mutations that alter substrate binding activity ofthe deaminase active site. In some embodiments, the cytidine deaminasecomprises one or more mutations that alter the conformation of DNA to berecognized and bound by the deaminase active site. In some embodiments,the cytidine deaminase comprises one or more mutations that alter thesubstrate accessibility to the deaminase active site. In someembodiments, arginine at residue 126 (R126) of APOBEC1 or acorresponding arginine residue in a homologous sequence is mutated. Insome embodiments, the catalytically inactive Cas13 is fused to or linkedto an APOBEC1 that comprises a R126A or R126E mutation. In someembodiments, tryptophan at residue 320 (R320) of APOBEC3G, or acorresponding arginine residue in a homologous sequence is mutated. Insome embodiments, the catalytically inactive Cas13 is fused to or linkedto an APOBEC3G mutant that comprises a R320A or R320E mutation. In someembodiments, arginine at residue 132 (R132) of APOBEC1 or acorresponding arginine residue in a homologous sequence is mutated. Insome embodiments, the catalytically inactive Cas13 is fused to or linkedto an APOBEC1 mutant that comprises a R132E mutation.

In some embodiments, the APOBEC1 domain of the CD-functionalized CRISPRsystem comprises one, two, or three mutations selected from W90Y, W90F,R126A, R126E, and R132E. In some embodiments, the APOBEC1 domaincomprises double mutations of W90Y and R126E. In some embodiments, theAPOBEC1 domain comprises double mutations of W90Y and R132E. In someembodiments, the APOBEC1 domain comprises double mutations of R126E andR132E. In some embodiments, the APOBEC1 domain comprises three mutationsof W90Y, R126E and R132E.

In some embodiments, one or more mutations in the cytidine deaminase asdisclosed herein reduce the editing window width to about 2 nucleotides.In some embodiments, one or more mutations in the cytidine deaminase asdisclosed herein reduce the editing window width to about 1 nucleotide.In some embodiments, one or more mutations in the cytidine deaminase asdisclosed herein reduce the editing window width while only minimally ormodestly affecting the editing efficiency of the enzyme. In someembodiments, one or more mutations in the cytidine deaminase asdisclosed herein reduce the editing window width without reducing theediting efficiency of the enzyme. In some embodiments, one or moremutations in the cytidine deaminase as disclosed herein enablediscrimination of neighboring cytidine nucleotides, which would beotherwise edited with similar efficiency by the cytidine deaminase.

In some embodiments, the cytidine deaminase protein further comprises oris connected to one or more double-stranded RNA (dsRNA) binding motifs(dsRBMs) or domains (dsRBDs) for recognizing and binding todouble-stranded nucleic acid substrates. In some embodiments, theinteraction between the cytidine deaminase and the substrate is mediatedby one or more additional protein factor(s), including a CRISPR/CASprotein factor. In some embodiments, the interaction between thecytidine deaminase and the substrate is further mediated by one or morenucleic acid component(s), including a guide RNA.

According to the present invention, the substrate of the cytidinedeaminase is an DNA single strand bubble of a RNA duplex comprising aCytosine of interest, made accessible to the cytidine deaminase uponbinding of the guide molecule to its DNA target which then forms theCRISPR-Cas complex with the CRISPR-Cas enzyme, whereby the cytosinedeaminase is fused to or is capable of binding to one or more componentsof the CRISPR-Cas complex, i.e. the CRISPR-Cas enzyme and/or the guidemolecule. The particular features of the guide molecule and CRISPR-Casenzyme are detailed below.

RNA-Binding Proteins

In certain example embodiments, the RNA-binding protein or functionaldomain thereof comprises a RNA recognition motif. In some examples, theRNA-binding protein may be a Cas effector protein, such as Cas13effector protein. In some cases, the RNA-binding protein may be acatalytically inactive Cas effector protein, such as dCas13.

In certain example embodiments, the RNA-binding protein comprises a zincfinger motif RNA-binding proteins or functional domains thereof maycomprise a Cyst-Hist, Gag-knuckle, Treble-clet, Zinc ribbon, Zn2/Cys6class motif.

In certain example embodiments, the RNA-binding protein may comprise aPumilio homology domain.

In one aspect the present invention provides methods for targeteddeamination of adenine in RNA, more particularly in an RNA sequence ofinterest. According to the methods of the invention, the adenosinedeaminase (AD) protein is recruited specifically to the relevant Adeninein the RNA sequence of interest by a CRISPR-Cas complex which canspecifically bind to a target sequence. In order to achieve this, theadenosine deaminase protein can either be covalently linked to theCRISPR-Cas enzyme or be provided as a separate protein, but adapted soas to ensure recruitment thereof to the CRISPR-Cas complex.

In particular embodiments, of the methods of the present invention,recruitment of the adenosine deaminase to the target locus is ensured byfusing the adenosine deaminase or catalytic domain thereof to theCRISPR-Cas protein, which is a Cas13 protein. Methods of generating afusion protein from two separate proteins are known in the art andtypically involve the use of spacers or linkers. The Cas13 protein canbe fused to the adenosine deaminase protein or catalytic domain thereofon either the N- or C-terminal end thereof. In particular embodiments,the CRISPR-Cas protein is an inactive or dead Cas13 protein and islinked to the N-terminus of the deaminase protein or its catalyticdomain.

The term “linker” as used in reference to a fusion protein refers to amolecule which joins the proteins to form a fusion protein. Generally,such molecules have no specific biological activity other than to joinor to preserve some minimum distance or other spatial relationshipbetween the proteins. However, in certain embodiments, the linker may beselected to influence some property of the linker and/or the fusionprotein such as the folding, net charge, or hydrophobicity of thelinker. The linker (e.g., a non-nucleotide loop) can be of any length.In some embodiments, the linker has a length equivalent to about 0-16nucleotides. In some embodiments, the linker has a length equivalent toabout 0-8 nucleotides. In some embodiments, the linker has a lengthequivalent to about 0-4 nucleotides. In some embodiments, the linker hasa length equivalent to about 2 nucleotides. Example linker design isalso described in WO2011/008730.

Suitable linkers for use in the methods of the present invention arewell known to those of skill in the art and include, but are not limitedto, straight or branched-chain carbon linkers, heterocyclic carbonlinkers, or peptide linkers. However, as used herein the linker may alsobe a covalent bond (carbon-carbon bond or carbon-heteroatom bond). Inparticular embodiments, the linker is used to separate the CRISPR-Casprotein and the adenosine deaminase by a distance sufficient to ensurethat each protein retains its required functional property. Preferredpeptide linker sequences adopt a flexible extended conformation and donot exhibit a propensity for developing an ordered secondary structure.In certain embodiments, the linker can be a chemical moiety which can bemonomeric, dimeric, multimeric or polymeric. Preferably, the linkercomprises amino acids. Typical amino acids in flexible linkers includeGly, Asn and Ser. Accordingly, in particular embodiments, the linkercomprises a combination of one or more of Gly, Asn and Ser amino acids.Other near neutral amino acids, such as Thr and Ala, also may be used inthe linker sequence. Exemplary linkers are disclosed in Maratea et al.(1985), Gene 40: 39-46; Murphy et al. (1986) Proc. Nat'l. Acad. Sci. USA83: 8258-62; U.S. Pat. No. 4,935,233; and U.S. Pat. No. 4,751,180. Forexample, GlySer linkers GGS, GGGS or GSG can be used. GGS, GSG, GGGS orGGGGS linkers can be used in repeats of 3 (such as (GGS)₃ (SEQ ID NO:12, (GGGGS)₃)(SEQ ID NO: 1) or 5, 6, 7, 9 or even 12 or more, to providesuitable lengths. In particular embodiments, linkers such as(GGGGS)3)(SEQ ID NO: 1) are preferably used herein. (GGGGS)₆) (SEQ IDNO: 4) (GGGGS)₉) (SEQ ID NO: 7) or (GGGGS)₁₂)(SEQ ID NO: 13) maypreferably be used as alternatives. Other preferred alternatives are(GGGGS)1)(SEQ ID NO: 14), (GGGGS)₂)(SEQ ID NO: 15), (GGGGS)₄)(SEQ ID NO:2), (GGGGS)₅)(SEQ ID NO: 3), (GGGGS)₇)(SEQ ID NO: 5), (GGGGS)₈)(SEQ IDNO: 6), (GGGGS)₁₀)(SEQ ID NO:8), or (GGGGS)₁₁)(SEQ ID NO: 9). In yet afurther embodiment, LEPGEKPYKCPECGKSFSQSGALTRHQRTHTR (SEQ ID NO:11) isused as a linker. In yet an additional embodiment, the linker is XTENlinker. In particular embodiments, the CRISPR-cas protein is a Cas13protein and is linked to the deaminase protein or its catalytic domainby means of an LEPGEKPYKCPECGKSFSQSGALTRHQRTHTR (SEQ ID NO:11) linker.In further particular embodiments, the Cas13 protein is linkedC-terminally to the N-terminus of a deaminase protein or its catalyticdomain by means of an LEPGEKPYKCPECGKSFSQSGALTRHQRTHTR (SEQ ID NO:11)linker. In addition, N- and C-terminal NLSs can also function as linker(e.g., PKKKRKVEASSPKKRKVEAS (SEQ ID NO:16)).

In particular embodiments of the methods of the present invention, theadenosine deaminase protein or catalytic domain thereof is delivered tothe cell or expressed within the cell as a separate protein, but ismodified so as to be able to link to either the Cas13 protein or theguide molecule. In particular embodiments, this is ensured by the use oforthogonal RNA-binding protein or adaptor protein/aptamer combinationsthat exist within the diversity of bacteriophage coat proteins. Examplesof such coat proteins include but are not limited to: MS2, Qβ, F2, GA,fr, JP501, M12, R17, BZ13, JP34, JP500, KU1, M11, MX1, TW18, VK, SP, FI,ID2, NL95, TW19, AP205, ϕCb5, ϕCb8r, ϕCb12r, ϕCb23r, 7s and PRR1.Aptamers can be naturally occurring or synthetic oligonucleotides thathave been engineered through repeated rounds of in vitro selection orSELEX (systematic evolution of ligands by exponential enrichment) tobind to a specific target.

In particular embodiments of the methods and systems of the presentinvention, the guide molecule is provided with one or more distinct RNAloop(s) or distinct sequence(s) that can recruit an adaptor protein. Aguide molecule may be extended, without colliding with the Cas13 proteinby the insertion of distinct RNA loop(s) or distinct sequence(s) thatmay recruit adaptor proteins that can bind to the distinct RNA loop(s)or distinct sequence(s). Examples of modified guides and their use inrecruiting effector domains to the CRISPR-Cas complex are provided inKonermann (Nature 2015, 517(7536): 583-588). In particular embodiments,the aptamer is a minimal hairpin aptamer which selectively bindsdimerized MS2 bacteriophage coat proteins in mammalian cells and isintroduced into the guide molecule, such as in the stemloop and/or in atetraloop. In these embodiments, the adenosine deaminase protein isfused to MS2. The adenosine deaminase protein is then co-deliveredtogether with the CRISPR-Cas protein and corresponding guide RNA.

The term “AD-functionalized CRISPR system” as used here refers to anucleic acid targeting and editing system comprising (a) a CRISPR-Casprotein, more particularly a Cas13 protein which is catalyticallyinactive; (b) a guide molecule which comprises a guide sequence; and (c)an adenosine deaminase protein or catalytic domain thereof; wherein theadenosine deaminase protein or catalytic domain thereof is covalently ornon-covalently linked to the CRISPR-Cas protein or the guide molecule oris adapted to link thereto after delivery; wherein the guide sequence issubstantially complementary to the target sequence but comprises anon-pairing C corresponding to the A being targeted for deamination,resulting in an A-C mismatch in an RNA duplex formed by the guidesequence and the target sequence. For application in eukaryotic cells,the CRISPR-Cas protein and/or the adenosine deaminase are preferablyNLS-tagged.

In some embodiments, the components (a), (b) and (c) are delivered tothe cell as a ribonucleoprotein complex. The ribonucleoprotein complexcan be delivered via one or more lipid nanoparticles.

In some embodiments, the components (a), (b) and (c) are delivered tothe cell as one or more RNA molecules, such as one or more guide RNAsand one or more mRNA molecules encoding the CRISPR-Cas protein, theadenosine deaminase protein, and optionally the adaptor protein. The RNAmolecules can be delivered via one or more lipid nanoparticles.

In some embodiments, the components (a), (b) and (c) are delivered tothe cell as one or more DNA molecules. In some embodiments, the one ormore DNA molecules are comprised within one or more vectors such asviral vectors (e.g., AAV). In some embodiments, the one or more DNAmolecules comprise one or more regulatory elements operably configuredto express the CRISPR-Cas protein, the guide molecule, and the adenosinedeaminase protein or catalytic domain thereof, optionally wherein theone or more regulatory elements comprise inducible promoters.

In some embodiments, the CRISPR-Cas protein is a dead Cas13. In someembodiments, the dead Cas13 is a dead Cas13a protein which comprises oneor more mutations in the HEPN domain. In some embodiments, the deadCas13a comprises a mutation corresponding to R474A and R1046A inLeptotrichia wadei (LwaCas13a). In some embodiments, the dead Cas13 is adead Cas13b protein which comprises one or more of R116A, H121A, R1177A,H1182A of a Cas13b protein originating from Bergeyella zoohelcum ATCC43767 or amino acid positions corresponding thereto of a Cas13bortholog.

In some embodiments of the guide molecule is capable of hybridizing witha target sequence comprising the Adenine to be deaminated within an RNAsequence to form an RNA duplex which comprises a non-pairing Cytosineopposite to said Adenine. Upon RNA duplex formation, the guide moleculeforms a complex with the Cas13 protein and directs the complex to bindthe RNA polynucleotide at the target RNA sequence of interest. Detailson the aspect of the guide of the AD-functionalized CRISPR-Cas systemare provided herein below.

In some embodiments, a Cas13 guide RNA having a canonical length of,e.g. LawCas13 is used to form an RNA duplex with the target DNA. In someembodiments, a Cas13 guide molecule longer than the canonical lengthfor, e.g. LawCas13a is used to form an RNA duplex with the target DNAincluding outside of the Cas13-guide RNA-target DNA complex.

In at least a first design, the AD-functionalized CRISPR systemcomprises (a) an adenosine deaminase fused or linked to a CRISPR-Casprotein, wherein the CRISPR-Cas protein is catalytically inactive, and(b) a guide molecule comprising a guide sequence designed to introducean A-C mismatch in an RNA duplex formed between the guide sequence andthe target sequence. In some embodiments, the CRISPR-Cas protein and/orthe adenosine deaminase are NLS-tagged, on either the N- or C-terminusor both.

In at least a second design, the AD-functionalized CRISPR systemcomprises (a) a CRISPR-Cas protein that is catalytically inactive, (b) aguide molecule comprising a guide sequence designed to introduce an A-Cmismatch in an RNA duplex formed between the guide sequence and thetarget sequence, and an aptamer sequence (e.g., MS2 RNA motif or PP7 RNAmotif) capable of binding to an adaptor protein (e.g., MS2 coatingprotein or PP7 coat protein), and (c) an adenosine deaminase fused orlinked to an adaptor protein, wherein the binding of the aptamer and theadaptor protein recruits the adenosine deaminase to the RNA duplexformed between the guide sequence and the target sequence for targeteddeamination at the A of the A-C mismatch. In some embodiments, theadaptor protein and/or the adenosine deaminase are NLS-tagged, on eitherthe N- or C-terminus or both. The CRISPR-Cas protein can also beNLS-tagged.

In some embodiments, a Cas13 guide RNA having a canonical length (e.g.,about 15-30 nt) is used to form a RNA duplex with the target RNA. Insome embodiments, a Cas13 guide molecule longer than the canonicallength (e.g., >30 nt) is used to form a RNA duplex with the target RNAincluding outside of the Cas13-guide RNA-target RNA complex.

In at least a first design, the CD-functionalized CRISPR systemcomprises (a) a cytidine deaminase fused or linked to a CRISPR-Casprotein, wherein the CRISPR-Cas protein is catalytically inactive Cas13,and (b) a guide molecule comprising a guide sequence, optionallydesigned to either (A) be upstream or downstream of the Cytosine ofinterest or (B) introduce a C-AU mismatch in a RNA duplex formed betweenthe guide sequence and the target sequence. In some embodiments, theCRISPR-Cas protein and/or the cytidine deaminase are NES-tagged, oneither the N- or C-terminus or both.

In at least a second design, the CD-functionalized CRISPR systemcomprises (a) a CRISPR-Cas protein that is catalytically inactive Cas13,(b) a guide molecule comprising a guide sequence, optionally designed toeither (A) be upstream or downstream of the Cytosine of interest or (B)introduce a C-A/U mismatch in a RNA duplex formed between the guidesequence and the target sequence, and an aptamer sequence (e.g., MS2 RNAmotif or PP7 RNA motif) capable of binding to an adaptor protein (e.g.,MS2 coating protein or PP7 coat protein), and (c) a cytidine deaminasefused or linked to an adaptor protein, wherein the binding of theaptamer and the adaptor protein recruits the cytidine deaminase to theRNA duplex formed between the guide sequence and the target sequence fortargeted deamination, either at a C outside the target sequence or atthe C of the optional C-A/U mismatch. In some embodiments, the adaptorprotein and/or the cytidine deaminase are NES-tagged, on either the N-or C-terminus or both. The CRISPR-Cas protein can also be NES-tagged.

The use of different aptamers and corresponding adaptor proteins alsoallows orthogonal gene editing to be implemented. In one example inwhich adenosine deaminase are used in combination with cytidinedeaminase for orthogonal gene editing/deamination, sgRNA targetingdifferent loci are modified with distinct RNA loops in order to recruitMS2-adenosine deaminase and PP7-cytidine deaminase (or PP7-adenosinedeaminase and MS2-cytidine deaminase), respectively, resulting inorthogonal deamination of A or C at the target loci of interested,respectively. PP7 is the RNA-binding coat protein of the bacteriophagePseudomonas. Like MS2, it binds a specific RNA sequence and secondarystructure. The PP7 RNA-recognition motif is distinct from that of MS2.Consequently, PP7 and MS2 can be multiplexed to mediate distinct effectsat different genomic loci simultaneously. For example, an sgRNAtargeting locus A can be modified with MS2 loops, recruitingMS2-adenosine deaminase, while another sgRNA targeting locus B can bemodified with PP7 loops, recruiting PP7-cytidine deaminase. In the samecell, orthogonal, locus-specific modifications are thus realized. Thisprinciple can be extended to incorporate other orthogonal RNA-bindingproteins.

In at least a third design, the AD-functionalized CRISPR systemcomprises (a) an adenosine deaminase inserted into an internal loop orunstructured region of a CRISPR-Cas protein, wherein the CRISPR-Casprotein is catalytically inactive or a nickase, and (b) a guide moleculecomprising a guide sequence designed to introduce an A-C mismatch in anRNA duplex formed between the guide sequence and the target sequence.

CRISPR-Cas protein split sites that are suitable for insertion ofadenosine deaminase can be identified with the help of a crystalstructure. One can use the crystal structure of an ortholog if arelatively high degree of homology exists between the ortholog and theintended CRISPR-Cas protein.

The split position may be located within a region or loop. Preferably,the split position occurs where an interruption of the amino acidsequence does not result in the partial or full destruction of astructural feature (e.g. alpha-helixes or (3-sheets). Unstructuredregions (regions that did not show up in the crystal structure becausethese regions are not structured enough to be “frozen” in a crystal) areoften preferred options. The positions within the unstructured regionsor outside loops may not need to be exactly the numbers provided above,but may vary by, for example 1, 2, 3, 4, 5, 6, 7, 8, 9, or even 10 aminoacids either side of the position given above, depending on the size ofthe loop, so long as the split position still falls within anunstructured region of outside loop.

The systems described herein can be used to target a specific Adeninewithin an RNA polynucleotide sequence for deamination. For example, theguide molecule can form a complex with the CRISPR-Cas protein anddirects the complex to bind a target RNA sequence in the RNApolynucleotide of interest. Because the guide sequence is designed tohave a non-pairing C, the RNA duplex formed between the guide sequenceand the target sequence comprises an A-C mismatch, which directs theadenosine deaminase to contact and deaminate the A opposite to thenon-pairing C, converting it to a Inosine (I). Since Inosine (I) basepairs with C and functions like G in cellular process, the targeteddeamination of A described herein are useful for correction ofundesirable G-A and C-T mutations, as well as for obtaining desirableA-G and T-C mutations.

In some embodiments, the systems may be used for targeted deamination inan RNA polynucleotide molecule in vitro. In some embodiments, theAD-functionalized CRISPR system is used for targeted deamination in aDNA molecule within a cell. The cell can be a eukaryotic cell, such as aanimal cell, a mammalian cell, a human, or a plant cell.

The invention also relates to a method for treating or preventing adisease by the targeted deamination using the AD-functionalized CRISPRsystem, wherein the deamination of the A, which remedies a diseasecaused by transcripts containing a pathogenic G→A or C→T point mutation.Examples of disease that can be treated or prevented with the presentinvention include cancer, Meier-Gorlin syndrome, Seckel syndrome 4,Joubert syndrome 5, Leber congenital amaurosis 10; Charcot-Marie-Toothdisease, type 2; Charcot-Marie-Tooth disease, type 2; Usher syndrome,type 2C; Spinocerebellar ataxia 28; Spinocerebellar ataxia 28;Spinocerebellar ataxia 28; Long QT syndrome 2; Sjögren-Larsson syndrome;Hereditary fructosuria; Hereditary fructosuria; Neuroblastoma;Neuroblastoma; Kallmann syndrome 1; Kallmann syndrome 1; Kallmannsyndrome 1; Metachromatic leukodystrophy.

The invention also relates to a method for knocking-out or knocking-downan undesirable activity of a gene, wherein the deamination of the A atthe transcript of the gene results in a loss of function. For example,in one embodiment, the targeted deamination by the AD-functionalizedCRISPR system can cause a nonsense mutation resulting in a prematurestop codon in an endogenous gene. This may alter the expression of theendogenous gene and can lead to a desirable trait in the edited cell. Inanother embodiment, the targeted deamination by the AD-functionalizedCRISPR system can cause a nonconservative missense mutation resulting ina code for a different amino acid residue in an endogenous gene. Thismay alter the function of the endogenous gene expressed and can alsolead to a desirable trait in the edited cell.

The invention also relates to a modified cell obtained by the targeteddeamination using the AD-functionalized CRISPR system, or progenythereof, wherein the modified cell comprises an I or G in replace of theA in the target RNA sequence of interest compared to a correspondingcell before the targeted deamination. The modified cell can be aeukaryotic cell, such as an animal cell, a plant cell, an mammaliancell, or a human cell.

In some embodiments, the modified cell is a therapeutic T cell, such asa T cell suitable for CAR-T therapies. The modification may result inone or more desirable traits in the therapeutic T cell, including butnot limited to, reduced expression of an immune checkpoint receptor(e.g., PDA, CTLA4), reduced expression of HLA proteins (e.g., B2M,HLA-A), and reduced expression of an endogenous TCR.

In some embodiments, the modified cell is an antibody-producing B cell.The modification may results in one or more desirable traits in the Bcell, including but not limited to, enhanced antibody production.

The invention also relates to a modified non-human animal or a modifiedplant. The modified non-human animal can be a farm animal. The modifiedplant can be an agricultural crop.

The invention further relates to a method for cell therapy, comprisingadministering to a patient in need thereof the modified cell describedherein, wherein the presence of the modified cell remedies a disease inthe patient. In one embodiment, the modified cell for cell therapy is aCAR-T cell capable of recognizing and/or attacking a tumor cell. Inanother embodiment, the modified cell for cell therapy is a stem cell,such as a neural stem cell, a mesenchymal stem cell, a hematopoieticstem cell, or an iPSC cell.

The invention additionally relates to an engineered, non-naturallyoccurring system suitable for modifying an Adenine in a target locus ofinterest, comprising: a guide molecule which comprises a guide sequence,or a nucleotide sequence encoding the guide molecule; a CRISPR-Casprotein, or one or more nucleotide sequences encoding the CRISPR-Casprotein; an adenosine deaminase protein or catalytic domain thereof, orone or more nucleotide sequences encoding; wherein the adenosinedeaminase protein or catalytic domain thereof is covalently ornon-covalently linked to the CRISPR-Cas protein or the guide molecule oris adapted to link thereto after delivery; wherein the guide sequence iscapable of hybridizing with a target sequence comprising an Adeninewithin an RNA polynucleotide of interest, but comprises a Cytosine atthe position corresponding to the Adenine.

The invention additionally relates to an engineered, non-naturallyoccurring vector system suitable for modifying an Adenine in a targetlocus of interest, comprising one or more vectors comprising: a firstregulatory element operably linked to one or more nucleotide sequencesencoding a guide molecule which comprises a guide sequence; a secondregulatory element operably linked to a nucleotide sequence encoding aCRISPR-Cas protein; and optionally a nucleotide sequence encoding anadenosine deaminase protein or catalytic domain thereof which is undercontrol of the first or second regulatory element or operably linked toa third regulatory element; wherein, if the nucleotide sequence encodingan adenosine deaminase protein or catalytic domain thereof is operablylinked to a third regulatory element, the adenosine deaminase protein orcatalytic domain thereof is adapted to link to the guide molecule or theCrispr-Cas protein after expression; wherein the guide sequence iscapable of hybridizing with a target sequence comprising an Adeninewithin the target locus, but comprises a Cytosine at the positioncorresponding to the Adenine; wherein components (a), (b) and (c) arelocated on the same or different vectors of the system.

The invention additionally relates to in vitro, ex vivo or in vivo hostcell or cell line or progeny thereof comprising the engineered,non-naturally occurring system or vector system described herein. Thehost cell can be a eukaryotic cell, such as an animal cell, a plantcell, an mammalian cell, or a human cell.

Base Excision Repair Inhibitor

In some embodiments, the systems further comprises a base excisionrepair (BER) inhibitor. Without wishing to be bound by any particulartheory, cellular DNA-repair response to the presence of I:T pairing maybe responsible for a decrease in nucleobase editing efficiency in cells.Alkyladenine DNA glycosylase (also known as DNA-3-methyladenineglycosylase, 3-alkyladenine DNA glycosylase, or N-methylpurine DNAglycosylase) catalyzes removal of hypoxanthine from DNA in cells, whichmay initiate base excision repair, with reversion of the I:T pair to aA:T pair as outcome.

In some embodiments, the BER inhibitor is an inhibitor of alkyladenineDNA glycosylase. In some embodiments, the BER inhibitor is an inhibitorof human alkyladenine DNA glycosylase. In some embodiments, the BERinhibitor is a polypeptide inhibitor. In some embodiments, the BERinhibitor is a protein that binds hypoxanthine. In some embodiments, theBER inhibitor is a protein that binds hypoxanthine in DNA. In someembodiments, the BER inhibitor is a catalytically inactive alkyladenineDNA glycosylase protein or binding domain thereof. In some embodiments,the BER inhibitor is a catalytically inactive alkyladenine DNAglycosylase protein or binding domain thereof that does not excisehypoxanthine from the DNA. Other proteins that are capable of inhibiting(e.g., sterically blocking) an alkyladenine DNA glycosylasebase-excision repair enzyme are within the scope of this disclosure.Additionally, any proteins that block or inhibit base-excision repair asalso within the scope of this disclosure.

Without wishing to be bound by any particular theory, base excisionrepair may be inhibited by molecules that bind the edited strand, blockthe edited base, inhibit alkyladenine DNA glycosylase, inhibit baseexcision repair, protect the edited base, and/or promote fixing of thenon-edited strand. It is believed that the use of the BER inhibitordescribed herein can increase the editing efficiency of an adenosinedeaminase that is capable of catalyzing a A to I change.

Accordingly, in the first design of the AD-functionalized CRISPR systemdiscussed above, the CRISPR-Cas protein or the adenosine deaminase canbe fused to or linked to a BER inhibitor (e.g., an inhibitor ofalkyladenine DNA glycosylase). In some embodiments, the BER inhibitorcan be comprised in one of the following structures (nCas13=Cas13nickase; dCas13=dead Cas13): [AD]-[optionallinker]-[nCas13/dCas13]-[optional linker]-[BER inhibitor];[AD]-[optional linker]-[BER inhibitor]-[optionallinker]-[nCas13/dCas13]; [BER inhibitor]-[optionallinker]-[AD]-[optional linker]-[nCas13/dCas13]; [BERinhibitor]-[optional linker]-[nCas13/dCas13]-[optional linker]-[AD];[nCas13/dCas13]-[optional linker]-[AD]-[optional linker]-[BERinhibitor]; [nCas13/dCas13]-[optional linker]-[BER inhibitor]-[optionallinker]-[AD].

Similarly, in the second design of the AD-functionalized CRISPR systemdiscussed above, the CRISPR-Cas protein, the adenosine deaminase, or theadaptor protein can be fused to or linked to a BER inhibitor (e.g., aninhibitor of alkyladenine DNA glycosylase). In some embodiments, the BERinhibitor can be comprised in one of the following structures(nCas13=Cas13 nickase; dCas13=dead Cas13): [nCas13/dCas13]-[optionallinker]-[BER inhibitor]; [BER inhibitor]-[optionallinker]-[nCas13/dCas13]; [AD]-[optional linker]-[Adaptor]-[optionallinker]-[BER inhibitor]; [AD]-[optional linker]-[BERinhibitor]-[optional linker]-[Adaptor]; [BER inhibitor]-[optionallinker]-[AD]-[optional linker]-[Adaptor]; [BER inhibitor]-[optionallinker]-[Adaptor]-[optional linker]-[AD]; [Adaptor]-[optionallinker]-[AD]-[optional linker]-[BER inhibitor]; [Adaptor]-[optionallinker]-[BER inhibitor]-[optional linker]-[AD].

In the third design of the AD-functionalized CRISPR system discussedabove, the BER inhibitor can be inserted into an internal loop orunstructured region of a CRISPR-Cas protein.

Targeting to the Nucleus

In some embodiments, the methods of the present invention relate tomodifying an Adenine in a target locus of interest, whereby the targetlocus is within a cell. In order to improve targeting of the CRISPR-Casprotein and/or the adenosine deaminase protein or catalytic domainthereof used in the methods of the present invention to the nucleus, itmay be advantageous to provide one or both of these components with oneor more nuclear localization sequences (NLSs).

In preferred embodiments, the NLSs used in the context of the presentinvention are heterologous to the proteins. Non-limiting examples ofNLSs include an NLS sequence derived from: the NLS of the SV40 viruslarge T-antigen, having the amino acid sequence PKKKRKV (SEQ ID NO: 17)or PKKKRKVEAS (SEQ ID NO: 18); the NLS from nucleoplasmin (e.g., thenucleoplasmin bipartite NLS with the sequence KRPAATKKAGQAKKKK (SEQ IDNO: 19)); the c-myc NLS having the amino acid sequence PAAKRVKLD (SEQ IDNO: 20) or RQRRNELKRSP (SEQ ID NO: 21); the hRNPA1 M9 NLS having thesequence NQSSNFGPMKGGNFGGRSSGPYGGGGQYFAKPRNQGGY (SEQ ID NO: 22); thesequence RMRIZFKNKGKDTAELRRRRVEVSVELRKAKKDEQILKRRNV (SEQ ID NO:23) ofthe IBB domain from importin-alpha; the sequences VSRKRPRP (SEQ IDNO:24) and PPKKARED (SEQ ID NO: 25) of the myoma T protein; the sequencePQPKKKPL (SEQ ID NO: 26) of human p53; the sequence SALIKKKKKMAP (SEQ IDNO: 27) of mouse c-abl IV; the sequences DRLRR (SEQ ID NO: 28) andPKQKKRK (SEQ ID NO: 29) of the influenza virus NS1; the sequenceRKLKKKIKKL (SEQ ID NO: 30) of the Hepatitis virus delta antigen; thesequence REKKKFLKRR (SEQ ID NO: 31) of the mouse Mx1 protein; thesequence KRKGDEVDGVDEVAKKKSKK (SEQ ID NO: 32) of the humanpoly(ADP-ribose) polymerase; and the sequence RKCLQAGMNLEARKTKK (SEQ IDNO: 33) of the steroid hormone receptors (human) glucocorticoid. Ingeneral, the one or more NLSs are of sufficient strength to driveaccumulation of the DNA-targeting Cas protein in a detectable amount inthe nucleus of a eukaryotic cell. In general, strength of nuclearlocalization activity may derive from the number of NLSs in theCRISPR-Cas protein, the particular NLS(s) used, or a combination ofthese factors. Detection of accumulation in the nucleus may be performedby any suitable technique. For example, a detectable marker may be fusedto the nucleic acid-targeting protein, such that location within a cellmay be visualized, such as in combination with a means for detecting thelocation of the nucleus (e.g., a stain specific for the nucleus such asDAPI). Cell nuclei may also be isolated from cells, the contents ofwhich may then be analyzed by any suitable process for detectingprotein, such as immunohistochemistry, Western blot, or enzyme activityassay. Accumulation in the nucleus may also be determined indirectly,such as by an assay for the effect of nucleic acid-targeting complexformation (e.g., assay for deaminase activity) at the target sequence,or assay for altered gene expression activity affected by DNA-targetingcomplex formation and/or DNA-targeting), as compared to a control notexposed to the CRISPR-Cas protein and deaminase protein, or exposed to aCRISPR-Cas and/or deaminase protein lacking the one or more NLSs.

The CRISPR-Cas and/or adenosine deaminase proteins may be provided with1 or more, such as with, 2, 3, 4, 5, 6, 7, 8, 9, 10, or moreheterologous NLSs. In some embodiments, the proteins comprises about ormore than about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more NLSs at or nearthe amino-terminus, about or more than about 1, 2, 3, 4, 5, 6, 7, 8, 9,10, or more NLSs at or near the carboxy-terminus, or a combination ofthese (e.g., zero or at least one or more NLS at the amino-terminus andzero or at one or more NLS at the carboxy terminus). When more than oneNLS is present, each may be selected independently of the others, suchthat a single NLS may be present in more than one copy and/or incombination with one or more other NLSs present in one or more copies.In some embodiments, an NLS is considered near the N- or C-terminus whenthe nearest amino acid of the NLS is within about 1, 2, 3, 4, 5, 10, 15,20, 25, 30, 40, 50, or more amino acids along the polypeptide chain fromthe N- or C-terminus. In preferred embodiments of the CRISPR-Casproteins, an NLS attached to the C-terminal of the protein.

In certain embodiments of the methods provided herein, the CRISPR-Casprotein and the deaminase protein are delivered to the cell or expressedwithin the cell as separate proteins. In these embodiments, each of theCRISPR-Cas and deaminase protein can be provided with one or more NLSsas described herein. In certain embodiments, the CRISPR-Cas anddeaminase proteins are delivered to the cell or expressed with the cellas a fusion protein. In these embodiments one or both of the CRISPR-Casand deaminase protein is provided with one or more NLSs. Where theadenosine deaminase is fused to an adaptor protein (such as MS2) asdescribed above, the one or more NLS can be provided on the adaptorprotein, provided that this does not interfere with aptamer binding. Inparticular embodiments, the one or more NLS sequences may also functionas linker sequences between the adenosine deaminase and the CRISPR-Casprotein.

In certain embodiments, guides of the invention comprise specificbinding sites (e.g. aptamers) for adapter proteins, which may be linkedto or fused to an adenosine deaminase or catalytic domain thereof. Whensuch a guides forms a CRISPR complex (i.e. CRISPR-Cas protein binding toguide and target) the adapter proteins bind and, the adenosine deaminaseor catalytic domain thereof associated with the adapter protein ispositioned in a spatial orientation which is advantageous for theattributed function to be effective.

The skilled person will understand that modifications to the guide whichallow for binding of the adapter+adenosine deaminase, but not properpositioning of the adapter +adenosine deaminase (e.g. due to sterichindrance within the three dimensional structure of the CRISPR complex)are modifications which are not intended. The one or more modified guidemay be modified at the tetra loop, the stem loop 1, stem loop 2, or stemloop 3, as described herein, preferably at either the tetra loop or stemloop 2, and most preferably at both the tetra loop and stem loop 2.

Use of Orthogonal Catalytically Inactive CRISPR-Cas Proteins

In particular embodiments, the Cas13 nickase is used in combination withan orthogonal catalytically inactive CRISPR-Cas protein to increaseefficiency of said Cas13 nickase (as described in Chen et al. 2017,Nature Communications 8:14958; doi:10.1038/ncomms14958). Moreparticularly, the orthogonal catalytically inactive CRISPR-Cas proteinis characterized by a different PAM recognition site than the Cas13nickase used in the system and the corresponding guide sequence isselected to bind to a target sequence proximal to that of the Cas13nickase of the system. The orthogonal catalytically inactive CRISPR-Casprotein as used in the context of the present invention does not formpart of the system but merely functions to increase the efficiency ofsaid Cas13 nickase and is used in combination with a standard guidemolecule as described in the art for said CRISPR-Cas protein. Inparticular embodiments, said orthogonal catalytically inactiveCRISPR-Cas protein is a dead CRISPR-Cas protein, i.e. comprising one ormore mutations which abolishes the nuclease activity of said CRISPR-Casprotein. In particular embodiments, the catalytically inactiveorthogonal CRISPR-Cas protein is provided with two or more guidemolecules which are capable of hybridizing to target sequences which areproximal to the target sequence of the Cas13 nickase. In particularembodiments, at least two guide molecules are used to target saidcatalytically inactive CRISPR-Cas protein, of which at least one guidemolecule is capable of hybridizing to a target sequence 5″ of the targetsequence of the Cas13 nickase and at least one guide molecule is capableof hybridizing to a target sequence 3′ of the target sequence of theCas13 nickase of the System, whereby said one or more target sequencesmay be on the same or the opposite DNA strand as the target sequence ofthe Cas13 nickase. In particular embodiments, the guide sequences forthe one or more guide molecules of the orthogonal catalytically inactiveCRISPR-Cas protein are selected such that the target sequences areproximal to that of the guide molecule for the targeting of theAD-functionalized CRISPR, i.e. for the targeting of the Cas13 nickase.In particular embodiments, the one or more target sequences of theorthogonal catalytically inactive CRISPR-Cas enzyme are each separatedfrom the target sequence of the Cas13 nickase by more than 5 but lessthan 450 basepairs. Optimal distances between the target sequences ofthe guides for use with the orthogonal catalytically inactive CRISPR-Casprotein and the target sequence of the System can be determined by theskilled person. In particular embodiments, the orthogonal CRISPR-Casprotein is a Class II, type II CRISPR protein. In particularembodiments, the orthogonal CRISPR-Cas protein is a Class II, type VCRISPR protein. In particular embodiments, the catalytically inactiveorthogonal CRISPR-Cas protein In particular embodiments, thecatalytically inactive orthogonal CRISPR-Cas protein has been modifiedto alter its PAM specificity as described elsewhere herein. Inparticular embodiments, the Cas13 protein nickase is a nickase which, byitself has limited activity in human cells, but which, in combinationwith an inactive orthogonal CRISPR-Cas protein and one or morecorresponding proximal guides ensures the required nickase activity.

Cas13 Effector Protein Complexes Can Deliver Functional Effectors

Unlike CRISPR-Cas13-mediated knockout, which eliminates expression bymutating at the RNA level, CRISPR-Cas13 knockdown allows for temporaryreduction of gene expression through the use of artificial transcriptionfactors, e.g., via mutating residues in cleavage domain(s) of the Cas13protein results in the generation of a catalytically inactive Cas13protein. A catalytically inactive Cas13 complexes with a guide RNA orcrRNA and localizes to the RNA sequence specified by that guide RNA's orcrRNA's targeting domain, however, it does not cleave the target. Fusionof the inactive Cas13 protein to an effector domain, e.g., atranscription repression domain, enables recruitment of the effector toany site specified by the guide RNA.

In some embodiments, dCas13 may be used for delivering a transcriptionfactor or an active domain thereof. The dCas13 may be fused with thetranscription factor or its active domain. The resulting complex may bedelivered under the guidance of a nuclei acid, e.g., a guide sequence.

Split Proteins

It is noted that in this context, and more generally for the variousapplications as described herein, the use of a split version of the RNAtargeting effector protein can be envisaged. Indeed, this may not onlyallow increased specificity but may also be advantageous for delivery.The Cas13 (e.g., Cas13b) may be split in the sense that the two parts ofthe Cas13 enzyme substantially comprise a functioning Cas13. Ideally,the split should always be so that the catalytic domain(s) areunaffected. That Cas13c may function as a nuclease or it may be adead-Cas13c which is essentially an RNA-binding protein with very littleor no catalytic activity, due to typically mutation(s) in its catalyticdomains.

Each half of the split Cas13 may be fused to a dimerization partner. Bymeans of example, and without limitation, employing rapamycin sensitivedimerization domains, allows to generate a chemically inducible splitCas13 for temporal control of Cas13 activity. Cas13 can thus be renderedchemically inducible by being split into two fragments and thatrapamycin-sensitive dimerization domains may be used for controlledreassembly of the Cas13. The two parts of the split Cas13c can bethought of as the N′ terminal part and the C′ terminal part of the splitCas13c. The fusion is typically at the split point of the Cas13. Inother words, the C′ terminal of the N′ terminal part of the split Cas13cis fused to one of the dimer halves, whilst the N′ terminal of the C′terminal part is fused to the other dimer half.

The Cas13 does not have to be split in the sense that the break is newlycreated. The split point is typically designed in silico and cloned intothe constructs. Together, the two parts of the split Cas13c, the N′terminal and C′ terminal parts, form a full Cas13, comprising preferablyat least 70% or more of the wildtype amino acids (or nucleotidesencoding them), preferably at least 80% or more, preferably at least 90%or more, preferably at least 95% or more, and most preferably at least99% or more of the wildtype amino acids (or nucleotides encoding them).Some trimming may be possible, and mutants are envisaged. Non-functionaldomains may be removed entirely. What is important is that the two partsmay be brought together and that the desired Cas13c function is restoredor reconstituted. The dimer may be a homodimer or a heterodimer.

In some cases, a first and a second split Cas13 effector proteins may becapable of fusing to each other to form a catalytically active Cas13effector protein. The fusing may be inducible.

Optimized Functional RNA Targeting Systems

In an aspect the invention thus provides a system for specific deliveryof functional components to the RNA environment. This can be ensuredusing the CRISPR systems comprising the RNA targeting effector proteinsof the present invention which allow specific targeting of differentcomponents to RNA. More particularly such components include activatorsor repressors, such as activators or repressors of RNA translation,degradation, etc.

According to one aspect the invention provides non-naturally occurringor engineered composition comprising a guide RNA or crRNA comprising aguide sequence capable of hybridizing to a target sequence of interestin a cell, wherein the guide RNA or crRNA is modified by the insertionof one or more distinct RNA sequence(s) that bind an adaptor protein. Inparticular embodiments, the RNA sequences may bind to two or moreadaptor proteins (e.g. aptamers), and wherein each adaptor protein isassociated with one or more functional domains. When there is more thanone functional domain, the functional domains can be same or different,e.g., two of the same or two different activators or repressors. In anaspect the invention provides a herein-discussed composition, whereinthe one or more functional domains are attached to the RNA targetingenzyme so that upon binding to the target RNA the functional domain isin a spatial orientation allowing for the functional domain to functionin its attributed function; In an aspect the invention provides aherein-discussed composition, wherein the composition comprises aCRISPR-Cas13 complex having at least three functional domains, at leastone of which is associated with the RNA targeting enzyme and at leasttwo of which are associated with the gRNA or crRNA.

CRISPR Development and Use

The present invention may be further illustrated and extended based onaspects of CRISPR-Cas development and use as set forth in the followingarticles and particularly as relates to delivery of a CRISPR proteincomplex and uses of an RNA guided endonuclease in cells and organisms:

-   -   Multiplex genome engineering using CRISPR-Cas systems. Cong, L.,        Ran, F. A., Cox, D., Lin, S., Barretto, R., Habib, N., Hsu, P.        D., Wu, X., Jiang, W., Marraffini, L. A., & Zhang, F. Science        February 15; 339(6121):819-23 (2013);    -   RNA-guided editing of bacterial genomes using CRISPR-Cas        systems. Jiang W., Bikard D., Cox D., Zhang F, Marraffini L A.        Nat Biotechnol March; 31(3):233-9 (2013);    -   One-Step Generation of Mice Carrying Mutations in Multiple Genes        by CRISPR-Cas-Mediated Genome Engineering. Wang H., Yang H.,        Shivalila C S., Dawlaty M M., Cheng A W., Zhang F., Jaenisch R.        Cell May 9; 153(4):910-8 (2013);    -   Optical control of mammalian endogenous transcription and        epigenetic states. Konermann S, Brigham M D, Trevino A E, Hsu P        D, Heidenreich M, Cong L, Platt R J, Scott D A, Church G M,        Zhang F. Nature. August 22; 500(7463):472-6. doi:        10.1038/Nature12466. Epub 2013 Aug. 23 (2013);    -   Double Nicking by RNA-Guided CRISPR Cas9 for Enhanced Genome        Editing Specificity. Ran, F A., Hsu, P D., Lin, C Y.,        Gootenberg, J S., Konermann, S., Trevino, A E., Scott, D A.,        Inoue, A., Matoba, S., Zhang, Y., & Zhang, F. Cell August 28.        pii: S0092-8674(13)01015-5 (2013-A);    -   DNA targeting specificity of RNA-guided Cas9 nucleases. Hsu, P.,        Scott, D., Weinstein, J., Ran, F A., Konermann, S., Agarwala,        V., Li, Y., Fine, E., Wu, X., Shalem, O., Cradick, T J.,        Marraffini, L A., Bao, G., & Zhang, F. Nat Biotechnol        doi:10.1038/nbt.2647 (2013);    -   Genome engineering using the CRISPR-Cas9 system. Ran, F A., Hsu,        P D., Wright, J., Agarwala, V., Scott, D A., Zhang, F. Nature        Protocols November; 8(11):2281-308 (2013-B);    -   Genome-Scale CRISPR-Cas9 Knockout Screening in Human Cells.        Shalem, O., Sanjana, N E., Hartenian, E., Shi, X., Scott, D A.,        Mikkelson, T., Heckl, D., Ebert, B L., Root, D E., Doench, J G.,        Zhang, F. Science December 12. (2013);    -   Crystal structure of cas9 in complex with guide RNA and target        DNA. Nishimasu, H., Ran, F A., Hsu, P D., Konermann, S.,        Shehata, S I., Dohmae, N., Ishitani, R., Zhang, F., Nureki, O.        Cell February 27, 156(5):935-49 (2014);    -   Genome-wide binding of the CRISPR endonuclease Cas9 in mammalian        cells. Wu X., Scott D A., Kriz A J., Chiu A C., Hsu P D., Dadon        D B., Cheng A W., Trevino A E., Konermann S., Chen S., Jaenisch        R., Zhang F., Sharp P A. Nat Biotechnol. April 20. doi:        10.1038/nbt.2889 (2014);    -   CRISPR-Cas9 Knockin Mice for Genome Editing and Cancer Modeling.        Platt R J, Chen S, Zhou Y, Yim M J, Swiech L, Kempton H R,        Dahlman J E, Parnas O, Eisenhaure T M, Jovanovic M, Graham D B,        Jhunjhunwala S, Heidenreich M, Xavier R J, Langer R, Anderson D        G, Hacohen N, Regev A, Feng G, Sharp P A, Zhang F. Cell 159(2):        440-455 DOI: 10.1016/j.cell.2014.09.014(2014);    -   Development and Applications of CRISPR-Cas9 for Genome        Engineering, Hsu P D, Lander E S, Zhang F., Cell. June 5;        157(6):1262-78 (2014).    -   Genetic screens in human cells using the CRISPR-Cas9 system,        Wang T, Wei J J, Sabatini D M, Lander E S., Science. January 3;        343(6166): 80-84. doi:10.1126/science.1246981 (2014);    -   Rational design of highly active sgRNAs for CRISPR-Cas9-mediated        gene inactivation, Doench J G, Hartenian E, Graham D B, Tothova        Z, Hegde M, Smith I, Sullender M, Ebert B L, Xavier R J, Root D        E., (published online 3 Sep. 2014) Nat Biotechnol. December;        32(12):1262-7 (2014);    -   In vivo interrogation of gene function in the mammalian brain        using CRISPR-Cas9, Swiech L, Heidenreich M, Banerjee A, Habib N,        Li Y, Trombetta J, Sur M, Zhang F., (published online 19        Oct. 2014) Nat Biotechnol. January; 33(1):102-6 (2015);    -   Genome-scale transcriptional activation by an engineered        CRISPR-Cas9 complex, Konermann S, Brigham M D, Trevino A E,        Joung J, Abudayyeh O O, Barcena C, Hsu P D, Habib N, Gootenberg        J S, Nishimasu H, Nureki O, Zhang F., Nature. January 29;        517(7536):583-8 (2015).    -   A split-Cas9 architecture for inducible genome editing and        transcription modulation, Zetsche B, Volz S E, Zhang F.,        (published online 2 Feb. 2015) Nat Biotechnol. February;        33(2):139-42 (2015);    -   Genome-wide CRISPR Screen in a Mouse Model of Tumor Growth and        Metastasis, Chen S, Sanjana N E, Zheng K, Shalem O, Lee K, Shi        X, Scott D A, Song J, Pan J Q, Weissleder R, Lee H, Zhang F,        Sharp P A. Cell 160, 1246-1260, Mar. 12, 2015 (multiplex screen        in mouse), and    -   In vivo genome editing using Staphylococcus aureus Cas9, Ran F        A, Cong L, Yan W X, Scott D A, Gootenberg J S, Kriz A J, Zetsche        B, Shalem O, Wu X, Makarova K S, Koonin E V, Sharp P A, Zhang        F., (published online 1 Apr. 2015), Nature. April 9;        520(7546):186-91 (2015).    -   Shalem et al., “High-throughput functional genomics using        CRISPR-Cas9,” Nature Reviews Genetics 16, 299-311 (May 2015).    -   Xu et al., “Sequence determinants of improved CRISPR sgRNA        design,” Genome Research 25, 1147-1157 (August 2015).    -   Parnas et al., “A Genome-wide CRISPR Screen in Primary Immune        Cells to Dissect Regulatory Networks,” Cell 162, 675-686 (Jul.        30, 2015).    -   Ramanan et al., CRISPR-Cas9 cleavage of viral DNA efficiently        suppresses hepatitis B virus,” Scientific Reports 5:10833. doi:        10.1038/srep10833 (Jun. 2, 2015)    -   Nishimasu et al., Crystal Structure of Staphylococcus aureus        Cas9,” Cell 162, 1113-1126 (Aug. 27, 2015)    -   BCL11A enhancer dissection by Cas9-mediated in situ saturating        mutagenesis, Canver et al., Nature 527(7577):192-7 (Nov.        12, 2015) doi: 10.1038/nature15521. Epub 2015 Sep. 16.    -   Cas13 Is a Single RNA-Guided Endonuclease of a Class 2        CRISPR-Cas System, Zetsche et al., Cell 163, 759-71 (Sep. 25,        2015).    -   Discovery and Functional Characterization of Diverse Class 2        CRISPR-Cas Systems, Shmakov et al., Molecular Cell, 60(3),        385-397 doi: 10.1016/j.molce1.2015.10.008 Epub Oct. 22, 2015.    -   Rationally engineered Cas9 nucleases with improved specificity,        Slaymaker et al., Science 2016 Jan. 1 351(6268): 84-88 doi:        10.1126/science.aad5227. Epub 2015 Dec. 1.    -   Gao et al, “Engineered Cas13 Enzymes with Altered PAM        Specificities,” bioRxiv 091611; doi: dx.doi.org/10.1101/091611        (Dec. 4, 2016).    -   each of which is incorporated herein by reference, may be        considered in the practice of the instant invention, and        discussed briefly below:    -   Cong et al. engineered type II CRISPR-Cas systems for use in        eukaryotic cells based on both Streptococcus thermophilus Cas9        and also Streptococcus pyogenes Cas9 and demonstrated that Cas9        nucleases can be directed by short RNAs to induce precise        cleavage of DNA in human and mouse cells. Their study further        showed that Cas9 as converted into a nicking enzyme can be used        to facilitate homology-directed repair in eukaryotic cells with        minimal mutagenic activity. Additionally, their study        demonstrated that multiple guide sequences can be encoded into a        single CRISPR array to enable simultaneous editing of several at        endogenous genomic loci sites within the mammalian genome,        demonstrating easy programmability and wide applicability of the        RNA-guided nuclease technology. This ability to use RNA to        program sequence specific DNA cleavage in cells defined a new        class of genome engineering tools. These studies further showed        that other CRISPR loci are likely to be transplantable into        mammalian cells and can also mediate mammalian genome cleavage.        Importantly, it can be envisaged that several aspects of the        CRISPR-Cas system can be further improved to increase its        efficiency and versatility.    -   Jiang et al. used the clustered, regularly interspaced, short        palindromic repeats (CRISPR)-associated Cas9 endonuclease        complexed with dual-RNAs to introduce precise mutations in the        genomes of Streptococcus pneumoniae and Escherichia coli. The        approach relied on dual-RNA:Cas9-directed cleavage at the        targeted genomic site to kill unmutated cells and circumvents        the need for selectable markers or counter-selection systems.        The study reported reprogramming dual-RNA:Cas9 specificity by        changing the sequence of short CRISPR RNA (crRNA) to make        single- and multinucleotide changes carried on editing        templates. The study showed that simultaneous use of two crRNAs        enabled multiplex mutagenesis. Furthermore, when the approach        was used in combination with recombineering, in S. pneumoniae,        nearly 100% of cells that were recovered using the described        approach contained the desired mutation, and in E. coli, 65%        that were recovered contained the mutation.    -   Wang et al. (2013) used the CRISPR-Cas system for the one-step        generation of mice carrying mutations in multiple genes which        were traditionally generated in multiple steps by sequential        recombination in embryonic stem cells and/or time-consuming        intercrossing of mice with a single mutation. The CRISPR-Cas        system will greatly accelerate the in vivo study of functionally        redundant genes and of epistatic gene interactions.    -   Konermann et al. (2013) addressed the need in the art for        versatile and robust technologies that enable optical and        chemical modulation of DNA-binding domains based CRISPR Cas9        enzyme and also Transcriptional Activator Like Effectors    -   Ran et al. (2013-A) described an approach that combined a Cas9        nickase mutant with paired guide RNAs to introduce targeted        double-strand breaks. This addresses the issue of the Cas9        nuclease from the microbial CRISPR-Cas system being targeted to        specific genomic loci by a guide sequence, which can tolerate        certain mismatches to the DNA target and thereby promote        undesired off-target mutagenesis. Because individual nicks in        the genome are repaired with high fidelity, simultaneous nicking        via appropriately offset guide RNAs is required for        double-stranded breaks and extends the number of specifically        recognized bases for target cleavage. The authors demonstrated        that using paired nicking can reduce off-target activity by 50-        to 1,500-fold in cell lines and to facilitate gene knockout in        mouse zygotes without sacrificing on-target cleavage efficiency.        This versatile strategy enables a wide variety of genome editing        applications that require high specificity.    -   Hsu et al. (2013) characterized SpCas9 targeting specificity in        human cells to inform the selection of target sites and avoid        off-target effects. The study evaluated >700 guide RNA variants        and SpCas9-induced indel mutation levels at >100 predicted        genomic off-target loci in 293T and 293FT cells. The authors        that SpCas9 tolerates mismatches between guide RNA and target        DNA at different positions in a sequence-dependent manner,        sensitive to the number, position and distribution of        mismatches. The authors further showed that SpCas9-mediated        cleavage is unaffected by DNA methylation and that the dosage of        SpCas9 and guide RNA can be titrated to minimize off-target        modification. Additionally, to facilitate mammalian genome        engineering applications, the authors reported providing a        web-based software tool to guide the selection and validation of        target sequences as well as off-target analyses.    -   Ran et al. (2013-B) described a set of tools for Cas9-mediated        genome editing via non-homologous end joining (NHEJ) or        homology-directed repair (HDR) in mammalian cells, as well as        generation of modified cell lines for downstream functional        studies. To minimize off-target cleavage, the authors further        described a double-nicking strategy using the Cas9 nickase        mutant with paired guide RNAs. The protocol provided by the        authors experimentally derived guidelines for the selection of        target sites, evaluation of cleavage efficiency and analysis of        off-target activity. The studies showed that beginning with        target design, gene modifications can be achieved within as        little as 1-2 weeks, and modified clonal cell lines can be        derived within 2-3 weeks.    -   Shalem et al. described a new way to interrogate gene function        on a genome-wide scale. Their studies showed that delivery of a        genome-scale CRISPR-Cas9 knockout (GeCKO) library targeted        18,080 genes with 64,751 unique guide sequences enabled both        negative and positive selection screening in human cells. First,        the authors showed use of the GeCKO library to identify genes        essential for cell viability in cancer and pluripotent stem        cells. Next, in a melanoma model, the authors screened for genes        whose loss is involved in resistance to vemurafenib, a        therapeutic that inhibits mutant protein kinase BRAF. Their        studies showed that the highest-ranking candidates included        previously validated genes NF1 and MED12 as well as novel hits        NF2, CUL3, TADA2B, and TADA1. The authors observed a high level        of consistency between independent guide RNAs targeting the same        gene and a high rate of hit confirmation, and thus demonstrated        the promise of genome-scale screening with Cas9.    -   Nishimasu et al. reported the crystal structure of Streptococcus        pyogenes Cas9 in complex with sgRNA and its target DNA at 2.5 A°        resolution. The structure revealed a bilobed architecture        composed of target recognition and nuclease lobes, accommodating        the sgRNA:DNAn RNA duplex in a positively charged groove at        their interface. Whereas the recognition lobe is essential for        binding sgRNA and DNA, the nuclease lobe contains the HNH and        RuvC nuclease domains, which are properly positioned for        cleavage of the complementary and non-complementary strands of        the target DNA, respectively. The nuclease lobe also contains a        carboxyl-terminal domain responsible for the interaction with        the protospacer adjacent motif (PAM). This high-resolution        structure and accompanying functional analyses have revealed the        molecular mechanism of RNA-guided DNA targeting by Cas9, thus        paving the way for the rational design of new, versatile        genome-editing technologies.    -   Wu et al. mapped genome-wide binding sites of a catalytically        inactive Cas9 (dCas9) from Streptococcus pyogenes loaded with        single guide RNAs (sgRNAs) in mouse embryonic stem cells        (mESCs). The authors showed that each of the four sgRNAs tested        targets dCas9 to between tens and thousands of genomic sites,        frequently characterized by a 5-nucleotide seed region in the        sgRNA and an NGG protospacer adjacent motif (PAM). Chromatin        inaccessibility decreases dCas9 binding to other sites with        matching seed sequences; thus 70% of off-target sites are        associated with genes. The authors showed that targeted        sequencing of 295 dCas9 binding sites in mESCs transfected with        catalytically active Cas9 identified only one site mutated above        background levels. The authors proposed a two-state model for        Cas9 binding and cleavage, in which a seed match triggers        binding but extensive pairing with target DNA is required for        cleavage.    -   Platt et al. established a Cre-dependent Cas9 knockin mouse. The        authors demonstrated in vivo as well as ex vivo genome editing        using adeno-associated virus (AAV)-, lentivirus-, or        particle-mediated delivery of guide RNA in neurons, immune        cells, and endothelial cells.    -   Hsu et al. (2014) is a review article that discusses generally        CRISPR-Cas9 history from yogurt to genome editing, including        genetic screening of cells.    -   Wang et al. (2014) relates to a pooled, loss-of-function genetic        screening approach suitable for both positive and negative        selection that uses a genome-scale lentiviral single guide RNA        (sgRNA) library.    -   Doench et al. created a pool of sgRNAs, tiling across all        possible target sites of a panel of six endogenous mouse and        three endogenous human genes and quantitatively assessed their        ability to produce null alleles of their target gene by antibody        staining and flow cytometry. The authors showed that        optimization of the PAM improved activity and also provided an        on-line tool for designing sgRNAs.    -   Swiech et al. demonstrate that AAV-mediated SpCas9 genome        editing can enable reverse genetic studies of gene function in        the brain.    -   Konermann et al. (2015) discusses the ability to attach multiple        effector domains, e.g., transcriptional activator, functional        and epigenomic regulators at appropriate positions on the guide        such as stem or tetraloop with and without linkers.    -   Zetsche et al. demonstrates that the Cas9 enzyme can be split        into two and hence the assembly of Cas9 for activation can be        controlled.    -   Chen et al. relates to multiplex screening by demonstrating that        a genome-wide in vivo CRISPR-Cas9 screen in mice reveals genes        regulating lung metastasis.    -   Ran et al. (2015) relates to SaCas9 and its ability to edit        genomes and demonstrates that one cannot extrapolate from        biochemical assays.    -   Shalem et al. (2015) described ways in which catalytically        inactive Cas9 (dCas9) fusions are used to synthetically repress        (CRISPRi) or activate (CRISPRa) expression, showing. advances        using Cas9 for genome-scale screens, including arrayed and        pooled screens, knockout approaches that inactivate genomic loci        and strategies that modulate transcriptional activity.    -   Xu et al. (2015) assessed the DNA sequence features that        contribute to single guide RNA (sgRNA) efficiency in        CRISPR-based screens. The authors explored efficiency of        CRISPR-Cas9 knockout and nucleotide preference at the cleavage        site. The authors also found that the sequence preference for        CRISPRi/a is substantially different from that for CRISPR-Cas9        knockout.    -   Parnas et al. (2015) introduced genome-wide pooled CRISPR-Cas9        libraries into dendritic cells (DCs) to identify genes that        control the induction of tumor necrosis factor (Tnf) by        bacterial lipopolysaccharide (LPS). Known regulators of Tlr4        signaling and previously unknown candidates were identified and        classified into three functional modules with distinct effects        on the canonical responses to LPS.    -   Ramanan et al (2015) demonstrated cleavage of viral episomal DNA        (cccDNA) in infected cells. The HBV genome exists in the nuclei        of infected hepatocytes as a 3.2 kb double-stranded episomal DNA        species called covalently closed circular DNA (cccDNA), which is        a key component in the HBV life cycle whose replication is not        inhibited by current therapies. The authors showed that sgRNAs        specifically targeting highly conserved regions of HBV robustly        suppresses viral replication and depleted cccDNA.    -   Nishimasu et al. (2015) reported the crystal structures of        SaCas9 in complex with a single guide RNA (sgRNA) and its        double-stranded DNA targets, containing the 5′-TTGAAT-3′ PAM and        the 5′-TTGGGT-3′ PAM. A structural comparison of SaCas9 with        SpCas9 highlighted both structural conservation and divergence,        explaining their distinct PAM specificities and orthologous        sgRNA recognition.    -   Canver et al. (2015) demonstrated a CRISPR-Cas9-based functional        investigation of non-coding genomic elements. The authors we        developed pooled CRISPR-Cas9 guide RNA libraries to perform in        situ saturating mutagenesis of the human and mouse BCL11A        enhancers which revealed critical features of the enhancers.    -   Zetsche et al. (2015) reported characterization of Cas13, a        class 2 CRISPR nuclease from Francisella novicida U112 having        features distinct from Cas9. Cas13 is a single RNA-guided        endonuclease lacking tracrRNA, utilizes a T-rich        protospacer-adjacent motif, and cleaves DNA via a staggered DNA        double-stranded break.    -   Shmakov et al. (2015) reported three distinct Class 2 CRISPR-Cas        systems. Two system CRISPR enzymes (C2c1 and C2c3) contain        RuvC-like endonuclease domains distantly related to Cas13.        Unlike Cas13, C2c1 depends on both crRNA and tracrRNA for DNA        cleavage. The third enzyme (C2c2) contains two predicted HEPN        RNase domains and is tracrRNA independent.    -   Slaymaker et al (2016) reported the use of structure-guided        protein engineering to improve the specificity of Streptococcus        pyogenes Cas9 (SpCas9). The authors developed “enhanced        specificity” SpCas9 (eSpCas9) variants which maintained robust        on-target cleavage with reduced off-target effects.

The methods and tools provided herein are exemplified for Cas13, a typeII nuclease that does not make use of tracrRNA. Orthologs of Cas13 havebeen identified in different bacterial species as described herein.Further type II nucleases with similar properties can be identifiedusing methods described in the art (Shmakov et al. 2015, 60:385-397;Abudayeh et al. 2016, Science, 5; 353(6299)) . In particularembodiments, such methods for identifying novel CRISPR effector proteinsmay comprise the steps of selecting sequences from the database encodinga seed which identifies the presence of a CRISPR Cas locus, identifyingloci located within 10 kb of the seed comprising Open Reading Frames(ORFs) in the selected sequences, selecting therefrom loci comprisingORFs of which only a single ORF encodes a novel CRISPR effector havinggreater than 700 amino acids and no more than 90% homology to a knownCRISPR effector. In particular embodiments, the seed is a protein thatis common to the CRISPR-Cas system, such as Cas1. In furtherembodiments, the CRISPR array is used as a seed to identify new effectorproteins.

The effectiveness of the present invention has been demonstrated.Preassembled recombinant CRISPR-Cas13 complexes comprising Cas13 andcrRNA may be transfected, for example by electroporation, resulting inhigh mutation rates and absence of detectable off-target mutations. Hur,J. K. et al, Targeted mutagenesis in mice by electroporation of Cas13ribonucleoproteins, Nat Biotechnol. 2016 Jun 6. doi: 10.1038/nbt.3596.Genome-wide analyses shows that Cas13 is highly specific. By onemeasure, in vitro cleavage sites determined for Cas13 in human HEK293Tcells were significantly fewer that for SpCas9. Kim, D. et al.,Genome-wide analysis reveals specificities of Cas13 endonucleases inhuman cells, Nat Biotechnol. 2016 Jun. 6. doi: 10.1038/nbt.3609. Anefficient multiplexed system employing Cas13 has been demonstrated inDrosophila employing gRNAs processed from an array containing inventingtRNAs. Port, F. et al, Expansion of the CRISPR toolbox in an animal withtRNA-flanked Cas9 and Cas13 gRNAs. doi: dx.doi.org/10.1101/046417.

Also, “Dimeric CRISPR RNA-guided FokI nucleases for highly specificgenome editing”, Shengdar Q. Tsai, Nicolas Wyvekens, Cyd Khayter,Jennifer A. Foden, Vishal Thapar, Deepak Reyon, Mathew J. Goodwin,Martin J. Aryee, J. Keith Joung Nature Biotechnology 32(6): 569-77(2014), relates to dimeric RNA-guided FokI Nucleases that recognizeextended sequences and can edit endogenous genes with high efficienciesin human cells.

With respect to general information on CRISPR/Cas Systems, componentsthereof, and delivery of such components, including methods, materials,delivery vehicles, vectors, particles, and making and using thereof,including as to amounts and formulations, as well asCRISPR-Cas-expressing eukaryotic cells, CRISPR-Cas expressingeukaryotes, such as a mouse, reference is made to: U.S. Pat. Nos.8,999,641, 8,993,233, 8,697,359, 8,771,945, 8,795,965, 8,865,406,8,871,445, 8,889,356, 8,889,418, 8,895,308, 8,906,616, 8,932,814, and8,945,839; US Patent Publications US 2014-0310830 (U.S. application Ser.No. 14/105,031), US 2014-0287938 A1 (U.S. application Ser. No.14/213,991), US 2014-0273234 A1 (U.S. application Ser. No. 14/293,674),US2014-0273232 A1 (U.S. application Ser. No. 14/290,575), US2014-0273231 (U.S. application Ser. No. 14/259,420), US 2014-0256046 A1(U.S. application Ser. No. 14/226,274), US 2014-0248702 A1(U.S.application Ser. No. 14/258,458), US 2014-0242700 A1 (U.S. applicationSer. No. 14/222,930), US 2014-0242699 A1 (U.S. application Ser. No.14/183,512), US 2014-0242664 A1 (U.S. application Ser. No. 14/104,990),US 2014-0234972 A1 (U.S. application Ser. No. 14/183,471), US2014-0227787 A1 (U.S. application Ser. No. 14/256,912), US 2014-0189896A1 (U.S. application Ser. No. 14/105,035), US 2014-0186958 (U.S.application Ser. No. 14/105,017), US 2014-0186919 A1 (U.S. applicationSer. No. 14/104,977), US 2014-0186843 A1 (U.S. application Ser. No.14/104,900), US 2014-0179770 A1 (U.S. application Ser. No. 14/104,837)and US 2014-0179006 A1 (U.S. application Ser. No. 14/183,486), US2014-0170753 (U.S. application Ser. No. 14/183,429); US 2015-0184139(U.S. application Ser. No. 14/324,960); Ser. No. 14/054,414 EuropeanPatent Applications EP 2 771 468 (EP13818570.7), EP 2 764 103(EP13824232.6), and EP 2 784 162 (EP14170383.5); and PCT PatentPublications WO2014/093661 (PCT/US2013/074743), WO2014/093694(PCT/US2013/074790), WO2014/093595 (PCT/U52013/074611), WO2014/093718(PCT/US2013/074825), WO2014/093709 (PCT/US2013/074812), WO2014/093622(PCT/US2013/074667), WO2014/093635 (PCT/US2013/074691), WO2014/093655(PCT/US2013/074736), WO2014/093712 (PCT/US2013/074819), WO2014/093701(PCT/U52013/074800), WO2014/018423 (PCT/U52013/051418), WO2014/204723(PCT/U52014/041790), WO2014/204724 (PCT/U52014/041800), WO2014/204725(PCT/U52014/041803), WO2014/204726 (PCT/U52014/041804), WO2014/204727(PCT/U52014/041806), WO2014/204728 (PCT/U52014/041808), WO2014/204729(PCT/U52014/041809), WO2015/089351 (PCT/US2014/069897), WO2015/089354(PCT/US2014/069902), WO2015/089364 (PCT/US2014/069925), WO2015/089427(PCT/U52014/070068), WO2015/089462 (PCT/US2014/070127), WO2015/089419(PCT/U52014/070057), WO2015/089465 (PCT/US2014/070135), WO2015/089486(PCT/U52014/070175), WO2015/058052 (PCT/US2014/061077), WO2015/070083(PCT/US2014/064663), WO2015/089354 (PCT/US2014/069902), WO2015/089351(PCT/US2014/069897), WO2015/089364 (PCT/US2014/069925), WO2015/089427(PCT/U52014/070068), WO2015/089473 (PCT/U52014/070152), WO2015/089486(PCT/U52014/070175), WO2016/049258 (PCT/US2015/051830), WO2016/094867(PCT/U52015/065385), WO2016/094872 (PCT/US2015/065393), WO2016/094874(PCT/U52015/065396), WO2016/106244 (PCT/US2015/067177).

Mention is also made of U.S. application 62/180,709, 17 Jun. 15,PROTECTED GUIDE RNAS (PGRNAS); U.S. application 62/091,455, filed, 12Dec. 14, PROTECTED GUIDE RNAS (PGRNAS); U.S. application 62/096,708, 24Dec. 14, PROTECTED GUIDE RNAS (PGRNAS); U.S. applications 62/091,462, 12Dec. 14, 62/096,324, 23 Dec. 14, 62/180,681, 17 Jun. 2015, and62/237,496, 5 Oct. 2015, DEAD GUIDES FOR CRISPR TRANSCRIPTION FACTORS;U.S. application 62/091,456, 12 Dec. 14 and 62/180,692, 17 Jun. 2015,ESCORTED AND FUNCTIONALIZED GUIDES FOR CRISPR-CAS SYSTEMS; U.S.application 62/091,461, 12 Dec. 14, DELIVERY, USE AND THERAPEUTICAPPLICATIONS OF THE CRISPR-CAS SYSTEMS AND COMPOSITIONS FOR GENOMEEDITING AS TO HEMATOPOETIC STEM CELLS (HSCs); U.S. application62/094,903, 19 Dec. 14, UNBIASED IDENTIFICATION OF DOUBLE-STRAND BREAKSAND GENOMIC REARRANGEMENT BY GENOME-WISE INSERT CAPTURE SEQUENCING; U.S.application 62/096,761, 24 Dec. 14, ENGINEERING OF SYSTEMS, METHODS ANDOPTIMIZED ENZYME AND GUIDE SCAFFOLDS FOR SEQUENCE MANIPULATION; U.S.application 62/098,059, 30 Dec. 14, 62/181,641, 18 Jun. 2015, and62/181,667, 18 Jun. 2015, RNA-TARGETING SYSTEM; U.S. application62/096,656, 24 Dec. 14 and 62/181,151, 17 Jun. 2015, CRISPR HAVING ORASSOCIATED WITH DESTABILIZATION DOMAINS; U.S. application 62/096,697, 24Dec. 14, CRISPR HAVING OR ASSOCIATED WITH AAV; U.S. application62/098,158, 30 Dec. 14, ENGINEERED CRISPR COMPLEX INSERTIONAL TARGETINGSYSTEMS; U.S. application 62/151,052, 22 Apr. 15, CELLULAR TARGETING FOREXTRACELLULAR EXOSOMAL REPORTING; U.S. application 62/054,490, 24 Sep.14, DELIVERY, USE AND THERAPEUTIC APPLICATIONS OF THE CRISPR-CAS SYSTEMSAND COMPOSITIONS FOR TARGETING DISORDERS AND DISEASES USING PARTICLEDELIVERY COMPONENTS; U.S. application 61/939,154, 12 Feb. 14, SYSTEMS,METHODS AND COMPOSITIONS FOR SEQUENCE MANIPULATION WITH OPTIMIZEDFUNCTIONAL CRISPR-CAS SYSTEMS; U.S. application 62/055,484, 25 Sep. 14,SYSTEMS, METHODS AND COMPOSITIONS FOR SEQUENCE MANIPULATION WITHOPTIMIZED FUNCTIONAL CRISPR-CAS SYSTEMS; U.S. application 62/087,537, 4Dec. 14, SYSTEMS, METHODS AND COMPOSITIONS FOR SEQUENCE MANIPULATIONWITH OPTIMIZED FUNCTIONAL CRISPR-CAS SYSTEMS; U.S. application62/054,651, 24 Sep. 14, DELIVERY, USE AND THERAPEUTIC APPLICATIONS OFTHE CRISPR-CAS SYSTEMS AND COMPOSITIONS FOR MODELING COMPETITION OFMULTIPLE CANCER MUTATIONS IN VIVO; U.S. application 62/067,886, 23 Oct.14, DELIVERY, USE AND THERAPEUTIC APPLICATIONS OF THE CRISPR-CAS SYSTEMSAND COMPOSITIONS FOR MODELING COMPETITION OF MULTIPLE CANCER MUTATIONSIN VIVO; U.S. applications 62/054,675, 24 Sep. 14 and 62/181,002, 17Jun. 2015, DELIVERY, USE AND THERAPEUTIC APPLICATIONS OF THE CRISPR-CASSYSTEMS AND COMPOSITIONS IN NEURONAL CELLS/TISSUES; U.S. application62/054,528, 24 Sep. 14, DELIVERY, USE AND THERAPEUTIC APPLICATIONS OFTHE CRISPR-CAS SYSTEMS AND COMPOSITIONS IN IMMUNE DISEASES OR DISORDERS;U.S. application 62/055,454, 25 Sep. 14, DELIVERY, USE AND THERAPEUTICAPPLICATIONS OF THE CRISPR-CAS SYSTEMS AND COMPOSITIONS FOR TARGETINGDISORDERS AND DISEASES USING CELL PENETRATION PEPTIDES (CPP); U.S.application 62/055,460, 25 Sep. 14, MULTIFUNCTIONAL-CRISPR COMPLEXESAND/OR OPTIMIZED ENZYME LINKED FUNCTIONAL-CRISPR COMPLEXES; U.S.application 62/087,475, 4 Dec. 14 and 62/181,690, 18 Jun. 2015,FUNCTIONAL SCREENING WITH OPTIMIZED FUNCTIONAL CRISPR-CAS SYSTEMS; U.S.application 62/055,487, 25 Sep. 14, FUNCTIONAL SCREENING WITH OPTIMIZEDFUNCTIONAL CRISPR-CAS SYSTEMS; U.S. application 62/087,546, 4 Dec. 14and 62/181,687, 18 Jun. 2015, MULTIFUNCTIONAL CRISPR COMPLEXES AND/OROPTIMIZED ENZYME LINKED FUNCTIONAL-CRISPR COMPLEXES; and U.S.application 62/098,285, 30 Dec. 14, CRISPR MEDIATED IN VIVO MODELING ANDGENETIC SCREENING OF TUMOR GROWTH AND METASTASIS.

Mention is made of U.S. applications 62/181,659, 18 Jun. 2015 and62/207,318, 19 Aug. 2015, ENGINEERING AND OPTIMIZATION OF SYSTEMS,METHODS, ENZYME AND GUIDE SCAFFOLDS OF CAS9 ORTHOLOGS AND VARIANTS FORSEQUENCE MANIPULATION. Mention is made of U.S. applications 62/181,663,18 Jun. 2015 and 62/245,264, 22 Oct. 2015, NOVEL CRISPR ENZYMES ANDSYSTEMS, U.S. applications 62/181,675, 18 Jun. 2015, 62/285,349, 22 Oct.2015, 62/296,522, 17 Feb. 2016, and 62/320,231, 8 Apr. 2016, NOVELCRISPR ENZYMES AND SYSTEMS, U.S. application 62/232,067, 24 Sep. 2015,U.S. application Ser. No. 14/975,085, 18 Dec. 2015, European applicationNo. 16150428.7, U.S. application 62/205,733, 16 Aug. 2015, U.S.application 62/201,542, 5 Aug. 2015, U.S. application 62/193,507, 16Jul. 2015, and U.S. application 62/181,739, 18 Jun. 2015, each entitledNOVEL CRISPR ENZYMES AND SYSTEMS and of U.S. application 62/245,270, 22Oct. 2015, NOVEL CRISPR ENZYMES AND SYSTEMS. Mention is also made ofU.S. application 61/939,256, 12 Feb. 2014, and WO 2015/089473(PCT/US2014/070152), 12 Dec. 2014, each entitled ENGINEERING OF SYSTEMS,METHODS AND OPTIMIZED GUIDE COMPOSITIONS WITH NEW ARCHITECTURES FORSEQUENCE MANIPULATION. Mention is also made of PCT/US2015/045504, 15Aug. 2015, U.S. application 62/180,699, 17 Jun. 2015, and U.S.application 62/038,358, 17 Aug. 2014, each entitled GENOME EDITING USINGCAS9 NICKASES.

Each of these patents, patent publications, and applications, and alldocuments cited therein or during their prosecution (“appin citeddocuments”) and all documents cited or referenced in the appin citeddocuments, together with any instructions, descriptions, productspecifications, and product sheets for any products mentioned therein orin any document therein and incorporated by reference herein, are herebyincorporated herein by reference, and may be employed in the practice ofthe invention. All documents (e.g., these patents, patent publicationsand applications and the appin cited documents) are incorporated hereinby reference to the same extent as if each individual document wasspecifically and individually indicated to be incorporated by reference.

Use of RNA-Targeting Effector Protein in Modulating Cellular Status

In certain embodiments Cas13 in a complex with crRNA is activated uponbinding to target RNA and subsequently cleaves any nearby ssRNA targets(i.e. “collateral” or “bystander” effects). Cas13, once primed by thecognate target, can cleave other (non-complementary) RNA molecules. Suchpromiscuous RNA cleavage could potentially cause cellular toxicity, orotherwise affect cellular physiology or cell status.

Accordingly, in certain embodiments, the non-naturally occurring orengineered composition, vector system, or delivery systems as describedherein are used for or are for use in induction of cell dormancy. Incertain embodiments, the non-naturally occurring or engineeredcomposition, vector system, or delivery systems as described herein areused for or are for use in induction of cell cycle arrest. In certainembodiments, the non-naturally occurring or engineered composition,vector system, or delivery systems as described herein are used for orare for use in reduction of cell growth and/or cell proliferation. Incertain embodiments, the non-naturally occurring or engineeredcomposition, vector system, or delivery systems as described herein areused for or are for use in induction of cell anergy. In certainembodiments, the non-naturally occurring or engineered composition,vector system, or delivery systems as described herein are used for orare for use in induction of cell apoptosis. In certain embodiments, thenon-naturally occurring or engineered composition, vector system, ordelivery systems as described herein are used for or are for use ininduction of cell necrosis. In certain embodiments, the non-naturallyoccurring or engineered composition, vector system, or delivery systemsas described herein are used for or are for use in induction of celldeath. In certain embodiments, the non-naturally occurring or engineeredcomposition, vector system, or delivery systems as described herein areused for or are for use in induction of programmed cell death.

In certain embodiments, the invention relates to a method for inductionof cell dormancy comprising introducing or inducing the non-naturallyoccurring or engineered composition, vector system, or delivery systemsas described herein. In certain embodiments, the invention relates to amethod for induction of cell cycle arrest comprising introducing orinducing the non-naturally occurring or engineered composition, vectorsystem, or delivery systems as described herein. In certain embodiments,the invention relates to a method for reduction of cell growth and/orcell proliferation comprising introducing or inducing the non-naturallyoccurring or engineered composition, vector system, or delivery systemsas described herein. In certain embodiments, the invention relates to amethod for induction of cell anergy comprising introducing or inducingthe non-naturally occurring or engineered composition, vector system, ordelivery systems as described herein. In certain embodiments, theinvention relates to a method for induction of cell apoptosis comprisingintroducing or inducing the non-naturally occurring or engineeredcomposition, vector system, or delivery systems as described herein. Incertain embodiments, the invention relates to a method for induction ofcell necrosis comprising introducing or inducing the non-naturallyoccurring or engineered composition, vector system, or delivery systemsas described herein. In certain embodiments, the invention relates to amethod for induction of cell death comprising introducing or inducingthe non-naturally occurring or engineered composition, vector system, ordelivery systems as described herein. In certain embodiments, theinvention relates to a method for induction of programmed cell deathcomprising introducing or inducing the non-naturally occurring orengineered composition, vector system, or delivery systems as describedherein.

Determination of PAM

Determination of PAM can be ensured as follows. This experiment closelyparallels similar work in E. coli for the heterologous expression ofStCas9 (Sapranauskas, R. et al. Nucleic Acids Res 39, 9275-9282 (2011)).Applicants introduce a plasmid containing both a PAM and a resistancegene into the heterologous E. coli, and then plate on the correspondingantibiotic. If there is DNA cleavage of the plasmid, Applicants observeno viable colonies.

In further detail, the assay is as follows for a DNA target. Two E. colistrains are used in this assay. One carries a plasmid that encodes theendogenous effector protein locus from the bacterial strain. The otherstrain carries an empty plasmid (e.g. pACYC184, control strain). Allpossible 7 or 8 bp PAM sequences are presented on an antibioticresistance plasmid (pUC19 with ampicillin resistance gene). The PAM islocated next to the sequence of proto-spacer 1 (the DNA target to thefirst spacer in the endogenous effector protein locus). Two PAMlibraries were cloned. One has a 8 random bp 5′ of the proto-spacer(e.g. total of 65536 different PAM sequences=complexity). The otherlibrary has 7 random bp 3′ of the proto-spacer (e.g. total complexity is16384 different PAMs). Both libraries were cloned to have in average 500plasmids per possible PAM. Test strain and control strain weretransformed with 5′PAM and 3′PAM library in separate transformations andtransformed cells were plated separately on ampicillin plates.Recognition and subsequent cutting/interference with the plasmid rendersa cell vulnerable to ampicillin and prevents growth. Approximately 12 hafter transformation, all colonies formed by the test and controlstrains where harvested and plasmid DNA was isolated. Plasmid DNA wasused as template for PCR amplification and subsequent deep sequencing.Representation of all PAMs in the untransfomed libraries showed theexpected representation of PAMs in transformed cells. Representation ofall PAMs found in control strains showed the actual representation.Representation of all PAMs in test strain showed which PAMs are notrecognized by the enzyme and comparison to the control strain allowsextracting the sequence of the depleted PAM.

The following PAMs have been identified for certain wild-type Cas13orthologues: the Acidaminococcus sp. BV3L6 Cas13 (AsCas13),Lachnospiraceae bacterium ND2006 Cas13 (LbCas13) and Prevotella albensis(PaCas13) can cleave target sites preceded by a TTTV PAM, where V is A/Cor G, FnCas13p, can cleave sites preceded by TTN, where N is A/C/G or T.The Moraxella bovoculi AAX08_00205, Moraxella bovoculi AAX11_00205,Butyrivibrio sp. NC3005, Thiomicrospira sp. XS5, or Lachnospiraceaebacterium MA2020 PAM is 5′ TTN, where N is A/C/G or T. The natural PAMsequence is TTTV or BTTV, wherein B is T/C or G and V is A/C or G andthe effector protein is Moraxella lacunata Cas13.

Codon Optimized Nucleic Acid Sequences

Where the effector protein is to be administered as a nucleic acid, theapplication envisages the use of codon-optimized CRISPR-Cas type Vprotein, and more particularly Cas13-encoding nucleic acid sequences(and optionally protein sequences). An example of a codon optimizedsequence, is in this instance a sequence optimized for expression in aeukaryote, e.g., humans (i.e. being optimized for expression in humans),or for another eukaryote, animal or mammal as herein discussed; see,e.g., SaCas9 human codon optimized sequence in WO 2014/093622(PCT/US2013/074667) as an example of a codon optimized sequence (fromknowledge in the art and this disclosure, codon optimizing codingnucleic acid molecule(s), especially as to effector protein (e.g.,Cas13) is within the ambit of the skilled artisan). Whilst this ispreferred, it will be appreciated that other examples are possible andcodon optimization for a host species other than human, or for codonoptimization for specific organs is known. In some embodiments, anenzyme coding sequence encoding a DNA/RNA-targeting Cas protein is codonoptimized for expression in particular cells, such as eukaryotic cells.The eukaryotic cells may be those of or derived from a particularorganism, such as a plant or a mammal, including but not limited tohuman, or non-human eukaryote or animal or mammal as herein discussed,e.g., mouse, rat, rabbit, dog, livestock, or non-human mammal orprimate. In some embodiments, processes for modifying the germ linegenetic identity of human beings and/or processes for modifying thegenetic identity of animals which are likely to cause them sufferingwithout any substantial medical benefit to man or animal, and alsoanimals resulting from such processes, may be excluded. In general,codon optimization refers to a process of modifying a nucleic acidsequence for enhanced expression in the host cells of interest byreplacing at least one codon (e.g., about or more than about 1, 2, 3, 4,5, 10, 15, 20, 25, 50, or more codons) of the native sequence withcodons that are more frequently or most frequently used in the genes ofthat host cell while maintaining the native amino acid sequence. Variousspecies exhibit particular bias for certain codons of a particular aminoacid. Codon bias (differences in codon usage between organisms) oftencorrelates with the efficiency of translation of messenger RNA (mRNA),which is in turn believed to be dependent on, among other things, theproperties of the codons being translated and the availability ofparticular transfer RNA (tRNA) molecules. The predominance of selectedtRNAs in a cell is generally a reflection of the codons used mostfrequently in peptide synthesis. Accordingly, genes can be tailored foroptimal gene expression in a given organism based on codon optimization.Codon usage tables are readily available, for example, at the “CodonUsage Database” available at www.kazusa.orjp/codon/ and these tables canbe adapted in a number of ways. See Nakamura, Y., et al. “Codon usagetabulated from the international DNA sequence databases: status for theyear 2000” Nucl. Acids Res. 28:292 (2000). Computer algorithms for codonoptimizing a particular sequence for expression in a particular hostcell are also available, such as Gene Forge (Aptagen; Jacobus, Pa.), arealso available. In some embodiments, one or more codons (e.g., 1, 2, 3,4, 5, 10, 15, 20, 25, 50, or more, or all codons) in a sequence encodinga DNA/RNA-targeting Cas protein corresponds to the most frequently usedcodon for a particular amino acid. As to codon usage in yeast, referenceis made to the online Yeast Genome database available atwww.yeastgenome.org/community/codon_usage.shtml, or Codon selection inyeast, Bennetzen and Hall, J Biol Chem. 1982 Mar. 25; 257(6):3026-31. Asto codon usage in plants including algae, reference is made to Codonusage in higher plants, green algae, and cyanobacteria, Campbell andGowri, Plant Physiol. 1990 January; 92(1): 1-11.; as well as Codon usagein plant genes, Murray et al, Nucleic Acids Res. 1989 Jan. 25;17(2):477-98; or Selection on the codon bias of chloroplast and cyanellegenes in different plant and algal lineages, Morton B R, J Mol Evol.1998 April; 46(4):449-59.

In certain example embodiments, the CRISPR Cas protein is selected fromTable 2.

TABLE 2 C2c2 orthologue Code Multi Letter Leptotrichia shahii C2-2 Lsh Lwadei F0279 (Lw2) C2-3 Lw2 Listeria seeligeri C2-4 Lse Lachnospiraceaebacterium MA2020 C2-5 LbM Lachnospiraceae bacterium NK4A179 C2-6 LbNK179[Clostridium] aminophilum DSM 10710 C2-7 Ca Carnobacterium gallinarumDSM 4847 C2-8 Cg Carnobacterium gallinarum DSM 4847 C2-9 Cg2Paludibacter propionicigenes WB4 C2-10 Pp Listeria weihenstephanensisFSL R9-0317 C2-11 Lwei Listeriaceae bacterium FSL M6-0635 C2-12 LbFSLLeptotrichia wadei F0279 C2-13 Lw Rhodobacter capsulatus SB 1003 C2-14Rc Rhodobacter capsulatus R121 C2-15 Rc Rhodobacter capsulatus DE442C2-16 Rc

In certain example embodiments, the CRISPR effector protein is a Cas13aprotein selected from Table 3

TABLE 3 c2c2-5 1 Lachnospiraceae bacterium MA2020 (SEQ ID NO: 34) c2c2-62 Lachnospiraceae bacterium NK4A179 (SEQ ID NO: 35) c2c2-7 3[Clostridium] aminophilum DSM 10710 (SEQ ID NO: 36) c2c2-8 5Carnobacterium gallinarum DSM 4847 (SEQ ID NO: 37) c2c2-9 6Carnobacterium gallinarum DSM 4847 (SEQ ID NO: 38) c2c2-10 7Paludibacter propionicigenes WB4 (SEQ ID NO: 39) c2c2-11 9 Listeriaweihenstephanensis FSL R9-0317 (SEQ ID NO: 40) c2c2-12 10 Listeriaceaebacterium FSL M6-0635 = (SEQ ID NO: 41) Listeria newyorkensis FSLM6-0635 c2c2-13(SEQ ID NO: 42) 12 Leptotrichia wadei F0279 c2c2-14(SEQID NO: 43) 15 Rhodobacter capsulatus SB 1003 c2c2-15(SEQ ID NO: 44) 16Rhodobacter capsulatus R121 c2c2-16 17 Rhodobacter capsulatus DE442 (SEQID NO: 45) c2c2-2 (SEQ ID NO: 46) c2c2-3(SEQ ID NO: 47) L wadei (Lw2)c2c2-4 Listeria seeligeri (SEQ ID NO: 48) C2-17(SEQ ID NO: 49)Leptotrichia buccalis C-1013-b C2-18 Herbinix hemicellulosilytica (SEQID NO: 50) C2-19 [Eubacterium] rectale (SEQ ID NO: 51) C2-20Eubacteriaceae bacterium CHKCI004 (SEQ ID NO: 52) C2-21 Blautia sp.Marseille-P2398 (SEQ ID NO: 53) C2-22 Leptotrichia sp. oral taxon 879str. F0557 (SEQ ID NO: 54) C2-23 Lachnospiraceae bacterium NK4A144 (SEQID NO: 55) C2-24 Chloroflexus aggregans (SEQ ID NO: 56) C2-25 Demequinaaurantiaca (SEQ ID NO: 57) C2-26 Thalassospira sp. TSL5-1 (SEQ ID NO:58) C2-27 SAMN04487830_13920 (SEQ ID NO: 59) [Pseudobutyrivibrio sp.OR37] C2-28 SAMN02910398_00008 [Butyrivibrio sp. (SEQ ID NO: 60)YAB3001] C2-29 Blautia sp. Marseille-P2398 (SEQ ID NO: 61) C2-30Leptotrichia sp. Marseille-P3007 (SEQ ID NO: 62) C2-31 Bacteroides ihuae(SEQ ID NO: 63) C2-32 SAMN05216357_1045 (SEQ ID NO: 64)[Porphyromonadaceae bacterium KH3CP3RA] C2-33 Listeria riparia (SEQ IDNO: 65) C2-34 Insolitispirillum peregrinum (SEQ ID NO: 66)

In certain example embodiments, the CRISPR effector protein is a Cas13bprotein selected from Table 4.

TABLE 4 Bergeyella zoohelcum  1 (SEQ ID NO: 67) Prevotella intermedia  2(SEQ ID NO: 68) Prevotella buccae  3 (SEQ ID NO: 69) Porphyromonasgingivalis  4 (SEQ ID NO: 70) Bacteroides pyogenes  5 (SEQ ID NO: 71)Alistipes sp. ZOR0009  6 (SEQ ID NO: 72) Prevotella sp. MA2016  7a (SEQID NO: 73) Prevotella sp. MA2016  7b (SEQ ID NO: 74) Riemerellaanatipestifer  8 (SEQ ID NO: 75) Prevotella aurantiaca  9 (SEQ ID NO:76) Prevotella saccharolytica 10 (SEQ ID NO: 77) HMPREF9712_03108[Myroides odoratimimus 11 CCUG 10230] (SEQ ID NO: 78) Prevotellaintermedia 12 (SEQ ID NO: 79) Capnocytophaga canimorsus 13 (SEQ ID NO:80) Porphyromonas gulae 14 (SEQ ID NO: 81) Prevotella sp. P5-125 15 (SEQID NO: 82) Flavobacterium branchiophilum 16 (SEQ ID NO: 83) Myroidesodoratimimus 17 (SEQ ID NO: 84) Flavobacterium columnare 18 (SEQ ID NO:85) Porphyromonas gingivalis 19 (SEQ ID NO: 86) Porphyromonas sp.COT-052 OH4946 20 (SEQ ID NO: 87) Prevotella intermedia 21 (SEQ ID NO:88) PIN17_0200 [Prevotella intermedia 17] AFJ07523 (SEQ ID NO: 89)Prevotella intermedia BAU18623 (SEQ ID NO: 90) HMPREF6485_0083[Prevotella buccae EFU31981 ATCC 33574] (SEQ ID NO: 91) HMPREF9144_1146[Prevotella pallens EGQ18444 ATCC 700821] (SEQ ID NO: 92)HMPREF9714_02132 [Myroides odoratimimus EHO08761 CCUG 12901] (SEQ ID NO:93) HMPREF9711_00870 [Myroides odoratimimus EKB06014 CCUG 3837] (SEQ IDNO: 94) HMPREF9699_02005 [Bergeyella zoohelcum EKB54193 ATCC 43767] (SEQID NO: 95) HMPREF9151_01387 [Prevotella saccharolytica EKY00089 F0055](SEQ ID NO: 96) A343_1752 [Porphyromonas gingivalis JCVI EOA10535 SC001](SEQ ID NO: 97) HMPREF1981_03090 [Bacteroides pyogenes ERI81700 F0041](SEQ ID NO: 98) HMPREF1553_02065 [Porphyromonas gingivalis ERJ65637F0568] (SEQ ID NO: 99) HMPREF1988_01768 [Porphyromonas gingivalisERJ81987 F0185] (SEQ ID NO: 100) HMPREF1990_01800 [Porphyromonasgingivalis ERJ87335 W4087]] (SEQ ID NO: 101) M573_117042 [Prevotellaintermedia ZT]] KJJ86756 (SEQ ID NO: 102) A2033_10205 [Bacteroidetesbacterium OFX18020.1 GWA2_31_9]] (SEQ ID NO: 103) SAMN05421542_0666[Chryseobacterium SDI27289.1 jejuense]] (SEQ ID NO: 104)SAMN05444360_11366 [Chryseobacterium SHM52812.1 carnipullorum]] (SEQ IDNO: 105) SAMN05421786_1011119 [Chryseobacterium SIS70481.1 ureilyticum]](SEQ ID NO: 106) Prevotella buccae] WP_004343581 (SEQ ID NO: 107)Porphyromonas gingivalis] WP_005873511 (SEQ ID NO: 108) Porphyromonasgingivalis] WP_005874195 (SEQ ID NO: 109) Prevotella pallens]WP_006044833 (SEQ ID NO: 110) Myroides odoratimimus] WP_006261414 (SEQID NO: 111) Myroides odoratimimus] WP_006265509 (SEQ ID NO: 112)Prevotella sp. MSX73] WP_007412163 (SEQ ID NO: 113) Porphyromonasgingivalis] WP_012458414 (SEQ ID NO: 114) Paludibacter propionicigenes]WP_013446107 (SEQ ID NO: 115) Porphyromonas gingivalis] WP_013816155(SEQ ID NO: 116) Flavobacterium columnare] WP_014165541 (SEQ ID NO: 117)Psychroflexus torquis] WP_015024765 (SEQ ID NO: 118) Riemerellaanatipestifer] WP_015345620 (SEQ ID NO: 119) Prevotella pleuritidis]WP_021584635 (SEQ ID NO: 120) Porphyromonas gingivalis WP_021663197 (SEQID NO: 121) Porphyromonas gingivalis WP_021665475 (SEQ ID NO: 122)Porphyromonas gingivalis WP_021677657 (SEQ ID NO: 123) Porphyromonasgingivalis WP_021680012 (SEQ ID NO: 124) Porphyromonas gingivalisWP_023846767 (SEQ ID NO: 125) Prevotella falsenii WP_036884929 (SEQ IDNO: 126) Prevotella pleuritidis WP_036931485 (SEQ ID NO: 127)[Porphyromonas gingivalis WP_039417390 (SEQ ID NO: 128) Porphyromonasgulae WP_039418912 (SEQ ID NO: 129) Porphyromonas gulae WP_039419792(SEQ ID NO: 130) Porphyromonas gulae WP_039426176 (SEQ ID NO: 131)Porphyromonas gulae WP_039431778 (SEQ ID NO: 132) Porphyromonas gulaeWP_039437199 (SEQ ID NO: 133) Porphyromonas gulae WP_039442171 (SEQ IDNO: 134) Porphyromonas gulae WP_039445055 (SEQ ID NO: 135)Capnocytophaga cynodegmi WP_041989581 (SEQ ID NO: 136) Prevotella sp.P5-119 WP_042518169 (SEQ ID NO: 137) Prevotella sp. P4-76 WP_044072147(SEQ ID NO: 138) Prevotella sp. P5-60 WP_044074780 (SEQ ID NO: 139)Phaeodactylibacter xiamenensis WP_044218239 (SEQ ID NO: 140)Flavobacterium sp. 316 WP_045968377 (SEQ ID NO: 141) Porphyromonas gulaeWP_046201018 (SEQ ID NO: 142) WP_047431796 Chryseobacterium (SEQ ID NO:143) sp. YR477 Riemerella anatipestifer WP_049354263 (SEQ ID NO: 144)Porphyromonas gingivalis WP_052912312 (SEQ ID NO: 145) Porphyromonasgingivalis WP_058019250 (SEQ ID NO: 146) Flavobacterium columnareWP_060381855 (SEQ ID NO: 147) Porphyromonas gingivalis WP_061156470 (SEQID NO: 148) Porphyromonas gingivalis WP 061156637 (SEQ ID NO: 149)Riemerella anatipestifer WP_061710138 (SEQ ID NO: 150) Flavobacteriumcolumnare WP_063744070 (SEQ ID NO: 151) Riemerella anatipestiferWP_064970887 (SEQ ID NO: 152) Sinomicrobium oceani WP_072319476.1 (SEQID NO: 153) Reichenbachiella agariperforans WP_073124441.1 (SEQ ID NO:154)

In certain example embodiments, the CRISPR effector protein is a Cas13cprotein from Table 5.

TABLE 5 Fusobacterium necrophorum subsp. funduliforme ATCC 51357contig00003 (SEQ ID NO: 155) Fusobacterium necrophorum DJ-2 contig0065,whole genome shotgun sequence (SEQ ID NO: 156) Fusobacterium necrophorumBFTR-1 contig0068 (SEQ ID NO: 157) Fusobacterium necrophorum subsp.funduliforme 1_1_36S cont1.14 (SEQ ID NO: 158) Fusobacterium perfoetensATCC 29250 T364DRAFT_scaffold00009.9_C (SEQ ID NO: 159) Fusobacteriumulcerans ATCC 49185 cont2.38 (SEQ ID NO: 160) Anaerosalibacter sp. ND1genome assembly Anaerosalibacter massiliensis ND1 (SEQ ID NO: 161)

Other DNA-Binding Proteins

In certain example embodiments the DNA-binding proteins may beTranscription Activator-Lake Effectors (TALES). Example TALES suitablefor use in the context of the present invention include, but are notlimited to, those disclosed in U.S. Patent Application No. 2012/0270273,and WO/2013/082519.

Delivery

In some embodiments, the components of the AD-functionalized system maybe delivered in various form, such as combinations of DNA/RNA or RNA/RNAor protein RNA. For example, the Cas13 protein may be delivered as aDNA-coding polynucleotide or an RNA-coding polynucleotide or as aprotein. The guide may be delivered may be delivered as a DNA-codingpolynucleotide or an RNA. All possible combinations are envisioned,including mixed forms of delivery.

In some aspects, the invention provides methods comprising deliveringone or more polynucleotides, such as or one or more vectors as describedherein, one or more transcripts thereof, and/or one or proteinstranscribed therefrom, to a host cell.

Vectors

In general, the term “vector” refers to a nucleic acid molecule capableof transporting another nucleic acid to which it has been linked. It isa replicon, such as a plasmid, phage, or cosmid, into which another DNAsegment may be inserted so as to bring about the replication of theinserted segment. Generally, a vector is capable of replication whenassociated with the proper control elementsVectors include, but are notlimited to, nucleic acid molecules that are single-stranded,double-stranded, or partially double-stranded; nucleic acid moleculesthat comprise one or more free ends, no free ends (e.g., circular);nucleic acid molecules that comprise DNA, RNA, or both; and othervarieties of polynucleotides known in the art. One type of vector is a“plasmid,” which refers to a circular double stranded DNA loop intowhich additional DNA segments can be inserted, such as by standardmolecular cloning techniques. Another type of vector is a viral vector,wherein virally-derived DNA or RNA sequences are present in the vectorfor packaging into a virus (e.g., retroviruses, replication defectiveretroviruses, adenoviruses, replication defective adenoviruses, andadeno-associated viruses). Viral vectors also include polynucleotidescarried by a virus for transfection into a host cell. Certain vectorsare capable of autonomous replication in a host cell into which they areintroduced (e.g., bacterial vectors having a bacterial origin ofreplication and episomal mammalian vectors). Other vectors (e.g.,non-episomal mammalian vectors) are integrated into the genome of a hostcell upon introduction into the host cell, and thereby are replicatedalong with the host genome. Moreover, certain vectors are capable ofdirecting the expression of genes to which they are operatively-linked.Such vectors are referred to herein as “expression vectors.” Vectors forand that result in expression in a eukaryotic cell can be referred toherein as “eukaryotic expression vectors.” Common expression vectors ofutility in recombinant DNA techniques are often in the form of plasmids.

Recombinant expression vectors can comprise a nucleic acid of theinvention in a form suitable for expression of the nucleic acid in ahost cell, which means that the recombinant expression vectors includeone or more regulatory elements, which may be selected on the basis ofthe host cells to be used for expression, that is operatively-linked tothe nucleic acid sequence to be expressed. Within a recombinantexpression vector, “operably linked” is intended to mean that thenucleotide sequence of interest is linked to the regulatory element(s)in a manner that allows for expression of the nucleotide sequence (e.g.,in an in vitro transcription/translation system or in a host cell whenthe vector is introduced into the host cell). Advantageous vectorsinclude lentiviruses and adeno-associated viruses, and types of suchvectors can also be selected for targeting particular types of cells.

With regards to recombination and cloning methods, mention is made ofU.S. patent application Ser. No. 10/815,730, published Sep. 2, 2004 asUS 2004-0171156 A1, the contents of which are herein incorporated byreference in their entirety.

The term “regulatory element” is intended to include promoters,enhancers, internal ribosomal entry sites (IRES), and other expressioncontrol elements (e.g., transcription termination signals, such aspolyadenylation signals and poly-U sequences). Such regulatory elementsare described, for example, in Goeddel, GENE EXPRESSION TECHNOLOGY:METHODS IN ENZYMOLOGY 185, Academic Press, San Diego, Calif. (1990).Regulatory elements include those that direct constitutive expression ofa nucleotide sequence in many types of host cell and those that directexpression of the nucleotide sequence only in certain host cells (e.g.,tissue-specific regulatory sequences). A tissue-specific promoter maydirect expression primarily in a desired tissue of interest, such asmuscle, neuron, bone, skin, blood, specific organs (e.g., liver,pancreas), or particular cell types (e.g., lymphocytes). Regulatoryelements may also direct expression in a temporal-dependent manner, suchas in a cell-cycle dependent or developmental stage-dependent manner,which may or may not also be tissue or cell-type specific. In someembodiments, a vector comprises one or more pol III promoter (e.g., 1,2, 3, 4, 5, or more pol III promoters), one or more pol II promoters(e.g., 1, 2, 3, 4, 5, or more pol II promoters), one or more pol Ipromoters (e.g., 1, 2, 3, 4, 5, or more pol I promoters), orcombinations thereof. Examples of pol III promoters include, but are notlimited to, U6 and H1 promoters. Examples of pol II promoters include,but are not limited to, the retroviral Rous sarcoma virus (RSV) LTRpromoter (optionally with the RSV enhancer), the cytomegalovirus (CMV)promoter (optionally with the CMV enhancer) [see, e.g., Boshart et al,Cell, 41:521-530 (1985)], the SV40 promoter, the dihydrofolate reductasepromoter, the β-actin promoter, the phosphoglycerol kinase (PGK)promoter, and the EF1α promoter. Also encompassed by the term“regulatory element” are enhancer elements, such as WPRE; CMV enhancers;the R-U5′ segment in LTR of HTLV-I (Mol. Cell. Biol., Vol. 8(1), p.466-472, 1988); SV40 enhancer; and the intron sequence between exons 2and 3 of rabbit β-globin (Proc. Natl. Acad. Sci. USA., Vol. 78(3), p.1527-31, 1981). It will be appreciated by those skilled in the art thatthe design of the expression vector can depend on such factors as thechoice of the host cell to be transformed, the level of expressiondesired, etc. A vector can be introduced into host cells to therebyproduce transcripts, proteins, or peptides, including fusion proteins orpeptides, encoded by nucleic acids as described herein (e.g., clusteredregularly interspersed short palindromic repeats (CRISPR) transcripts,proteins, enzymes, mutant forms thereof, fusion proteins thereof, etc.).With regards to regulatory sequences, mention is made of U.S. patentapplication Ser. No. 10/491,026, the contents of which are incorporatedby reference herein in their entirety. With regards to promoters,mention is made of PCT publication WO 2011/028929 and U.S. applicationSer. No. 12/511,940, the contents of which are incorporated by referenceherein in their entirety.

Advantageous vectors include lentiviruses and adeno-associated viruses,and types of such vectors can also be selected for targeting particulartypes of cells.

In particular embodiments, use is made of bicistronic vectors for theguide RNA and (optionally modified or mutated) the CRISPR-Cas proteinfused to adenosine deaminase. Bicistronic expression vectors for guideRNA and (optionally modified or mutated) CRISPR-Cas protein fused toadenosine deaminase are preferred. In general and particularly in thisembodiment, (optionally modified or mutated) CRISPR-Cas protein fused toadenosine deaminase is preferably driven by the CBh promoter. The RNAmay preferably be driven by a Pol III promoter, such as a U6 promoter.Ideally the two are combined.

Vectors can be designed for expression of CRISPR transcripts (e.g.nucleic acid transcripts, proteins, or enzymes) in prokaryotic oreukaryotic cells. For example, CRISPR transcripts can be expressed inbacterial cells such as Escherichia coli, insect cells (usingbaculovirus expression vectors), yeast cells, or mammalian cells.Suitable host cells are discussed further in Goeddel, GENE EXPRESSIONTECHNOLOGY: METHODS IN ENZYMOLOGY 185, Academic Press, San Diego, Calif.(1990). Alternatively, the recombinant expression vector can betranscribed and translated in vitro, for example using T7 promoterregulatory sequences and T7 polymerase.

Vectors may be introduced and propagated in a prokaryote or prokaryoticcell. In some embodiments, a prokaryote is used to amplify copies of avector to be introduced into a eukaryotic cell or as an intermediatevector in the production of a vector to be introduced into a eukaryoticcell (e.g. amplifying a plasmid as part of a viral vector packagingsystem). In some embodiments, a prokaryote is used to amplify copies ofa vector and express one or more nucleic acids, such as to provide asource of one or more proteins for delivery to a host cell or hostorganism. Expression of proteins in prokaryotes is most often carriedout in Escherichia coli with vectors containing constitutive orinducible promoters directing the expression of either fusion ornon-fusion proteins. Fusion vectors add a number of amino acids to aprotein encoded therein, such as to the amino terminus of therecombinant protein. Such fusion vectors may serve one or more purposes,such as: (i) to increase expression of recombinant protein; (ii) toincrease the solubility of the recombinant protein; and (iii) to aid inthe purification of the recombinant protein by acting as a ligand inaffinity purification. Often, in fusion expression vectors, aproteolytic cleavage site is introduced at the junction of the fusionmoiety and the recombinant protein to enable separation of therecombinant protein from the fusion moiety subsequent to purification ofthe fusion protein. Such enzymes, and their cognate recognitionsequences, include Factor Xa, thrombin and enterokinase. Example fusionexpression vectors include pGEX (Pharmacia Biotech Inc; Smith andJohnson, 1988. Gene 67: 31-40), pMAL (New England Biolabs, Beverly,Mass.) and pRIT5 (Pharmacia, Piscataway, N.J.) that fuse glutathioneS-transferase (GST), maltose E binding protein, or protein A,respectively, to the target recombinant protein. Examples of suitableinducible non-fusion E. coli expression vectors include pTrc (Amrann etal., (1988) Gene 69:301-315) and pET 11d (Studier et al., GENEEXPRESSION TECHNOLOGY: METHODS IN ENZYMOLOGY 185, Academic Press, SanDiego, Calif. (1990) 60-89). In some embodiments, a vector is a yeastexpression vector. Examples of vectors for expression in yeastSaccharomyces cerivisae include pYepSec1 (Baldari, et al., 1987. EMBO J.6: 229-234), pMFa (Kuijan and Herskowitz, 1982. Cell 30: 933-943),pJRY88 (Schultz et al., 1987. Gene 54: 113-123), pYES2 (InvitrogenCorporation, San Diego, Calif.), and picZ (InVitrogen Corp, San Diego,Calif.). In some embodiments, a vector drives protein expression ininsect cells using baculovirus expression vectors. Baculovirus vectorsavailable for expression of proteins in cultured insect cells (e.g., SF9cells) include the pAc series (Smith, et al., 1983. Mol. Cell. Biol. 3:2156-2165) and the pVL series (Lucklow and Summers, 1989. Virology 170:31-39).

In some embodiments, a vector is capable of driving expression of one ormore sequences in mammalian cells using a mammalian expression vector.Examples of mammalian expression vectors include pCDM8 (Seed, 1987.Nature 329: 840) and pMT2PC (Kaufman, et al., 1987. EMBO J. 6: 187-195).When used in mammalian cells, the expression vector's control functionsare typically provided by one or more regulatory elements. For example,commonly used promoters are derived from polyoma, adenovirus 2,cytomegalovirus, simian virus 40, and others disclosed herein and knownin the art. For other suitable expression systems for both prokaryoticand eukaryotic cells see, e.g., Chapters 16 and 17 of Sambrook, et al.,MOLECULAR CLONING: A LABORATORY MANUAL. 2nd ed., Cold Spring HarborLaboratory, Cold Spring Harbor Laboratory Press, Cold Spring Harbor,N.Y., 1989.

In some embodiments, the recombinant mammalian expression vector iscapable of directing expression of the nucleic acid preferentially in aparticular cell type (e.g., tissue-specific regulatory elements are usedto express the nucleic acid). Tissue-specific regulatory elements areknown in the art. Non-limiting examples of suitable tissue-specificpromoters include the albumin promoter (liver-specific; Pinkert, et al.,1987. Genes Dev. 1: 268-277), lymphoid-specific promoters (Calame andEaton, 1988. Adv. Immunol. 43: 235-275), in particular promoters of Tcell receptors (Winoto and Baltimore, 1989. EMBO J. 8: 729-733) andimmunoglobulins (Baneiji, et al., 1983. Cell 33: 729-740; Queen andBaltimore, 1983. Cell 33: 741-748), neuron-specific promoters (e.g., theneurofilament promoter; Byrne and Ruddle, 1989. Proc. Natl. Acad. Sci.USA 86: 5473-5477), pancreas-specific promoters (Edlund, et al., 1985.Science 230: 912-916), and mammary gland-specific promoters (e.g., milkwhey promoter; U.S. Pat. No. 4,873,316 and European ApplicationPublication No. 264,166). Developmentally-regulated promoters are alsoencompassed, e.g., the murine hox promoters (Kessel and Gruss, 1990.Science 249: 374-379) and the α-fetoprotein promoter (Campes andTilghman, 1989. Genes Dev. 3: 537-546). With regards to theseprokaryotic and eukaryotic vectors, mention is made of U.S. Pat. No.6,750,059, the contents of which are incorporated by reference herein intheir entirety. Other embodiments of the invention may relate to the useof viral vectors, with regards to which mention is made of U.S. patentapplication Ser. No. 13/092,085, the contents of which are incorporatedby reference herein in their entirety. Tissue-specific regulatoryelements are known in the art and in this regard, mention is made ofU.S. Pat. No. 7,776,321, the contents of which are incorporated byreference herein in their entirety. In some embodiments, a regulatoryelement is operably linked to one or more elements of a CRISPR system soas to drive expression of the one or more elements of the CRISPR system.

In some embodiments, one or more vectors driving expression of one ormore elements of a nucleic acid-targeting system are introduced into ahost cell such that expression of the elements of the nucleicacid-targeting system direct formation of a nucleic acid-targetingcomplex at one or more target sites. For example, a nucleicacid-targeting effector enzyme and a nucleic acid-targeting guide RNAcould each be operably linked to separate regulatory elements onseparate vectors. RNA(s) of the nucleic acid-targeting system can bedelivered to a transgenic nucleic acid-targeting effector protein animalor mammal, e.g., an animal or mammal that constitutively or inducibly orconditionally expresses nucleic acid-targeting effector protein; or ananimal or mammal that is otherwise expressing nucleic acid-targetingeffector proteins or has cells containing nucleic acid-targetingeffector proteins, such as by way of prior administration thereto of avector or vectors that code for and express in vivo nucleicacid-targeting effector proteins. Alternatively, two or more of theelements expressed from the same or different regulatory elements, maybe combined in a single vector, with one or more additional vectorsproviding any components of the nucleic acid-targeting system notincluded in the first vector. nucleic acid-targeting system elementsthat are combined in a single vector may be arranged in any suitableorientation, such as one element located 5′ with respect to (“upstream”of) or 3′ with respect to (“downstream” of) a second element. The codingsequence of one element may be located on the same or opposite strand ofthe coding sequence of a second element, and oriented in the same oropposite direction. In some embodiments, a single promoter drivesexpression of a transcript encoding a nucleic acid-targeting effectorprotein and the nucleic acid-targeting guide RNA, embedded within one ormore intron sequences (e.g., each in a different intron, two or more inat least one intron, or all in a single intron). In some embodiments,the nucleic acid-targeting effector protein and the nucleicacid-targeting guide RNA may be operably linked to and expressed fromthe same promoter. Delivery vehicles, vectors, particles, nanoparticles,formulations and components thereof for expression of one or moreelements of a nucleic acid-targeting system are as used in the foregoingdocuments, such as WO 2014/093622 (PCT/US2013/074667). In someembodiments, a vector comprises one or more insertion sites, such as arestriction endonuclease recognition sequence (also referred to as a“cloning site”). In some embodiments, one or more insertion sites (e.g.,about or more than about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or moreinsertion sites) are located upstream and/or downstream of one or moresequence elements of one or more vectors. When multiple different guidesequences are used, a single expression construct may be used to targetnucleic acid-targeting activity to multiple different, correspondingtarget sequences within a cell. For example, a single vector maycomprise about or more than about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20,or more guide sequences. In some embodiments, about or more than about1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more such guide-sequence-containingvectors may be provided, and optionally delivered to a cell. In someembodiments, a vector comprises a regulatory element operably linked toan enzyme-coding sequence encoding a a nucleic acid-targeting effectorprotein. Nucleic acid-targeting effector protein or nucleicacid-targeting guide RNA or RNA(s) can be delivered separately; andadvantageously at least one of these is delivered via a particlecomplex. nucleic acid-targeting effector protein mRNA can be deliveredprior to the nucleic acid-targeting guide RNA to give time for nucleicacid-targeting effector protein to be expressed. Nucleic acid-targetingeffector protein mRNA might be administered 1-12 hours (preferablyaround 2-6 hours) prior to the administration of nucleic acid-targetingguide RNA. Alternatively, nucleic acid-targeting effector protein mRNAand nucleic acid-targeting guide RNA can be administered together.Advantageously, a second booster dose of guide RNA can be administered1-12 hours (preferably around 2-6 hours) after the initialadministration of nucleic acid-targeting effector protein mRNA+guideRNA. Additional administrations of nucleic acid-targeting effectorprotein mRNA and/or guide RNA might be useful to achieve the mostefficient levels of genome modification.

Conventional viral and non-viral based gene transfer methods can be usedto introduce nucleic acids in mammalian cells or target tissues. Suchmethods can be used to administer nucleic acids encoding components of anucleic acid-targeting system to cells in culture, or in a hostorganism. Non-viral vector delivery systems include DNA plasmids, RNA(e.g. a transcript of a vector described herein), naked nucleic acid,and nucleic acid complexed with a delivery vehicle, such as a liposome.Viral vector delivery systems include DNA and RNA viruses, which haveeither episomal or integrated genomes after delivery to the cell. For areview of gene therapy procedures, see Anderson, Science 256:808-813(1992); Nabel & Felgner, TIBTECH 11:211-217 (1993); Mitani & Caskey,TIBTECH 11:162-166 (1993); Dillon, TIBTECH 11:167-175 (1993); Miller,Nature 357:455-460 (1992); Van Brunt, Biotechnology 6(10):1149-1154(1988); Vigne, Restorative Neurology and Neuroscience 8:35-36 (1995);Kremer & Perricaudet, British Medical Bulletin 51(1):31-44 (1995);Haddada et al., in Current Topics in Microbiology and Immunology,Doerfler and Bohm (eds) (1995); and Yu et al., Gene Therapy 1:13-26(1994).

Methods of non-viral delivery of nucleic acids include lipofection,nucleofection, microinjection, biolistics, virosomes, liposomes,immunoliposomes, polycation or lipid:nucleic acid conjugates, naked DNA,artificial virions, and agent-enhanced uptake of DNA. Lipofection isdescribed in e.g., U.S. Pat. Nos. 5,049,386, 4,946,787; and 4,897,355)and lipofection reagents are sold commercially (e.g., Transfectam™ andLipofectin™). Cationic and neutral lipids that are suitable forefficient receptor-recognition lipofection of polynucleotides includethose of Felgner, WO 91/17424; WO 91/16024. Delivery can be to cells(e.g. in vitro or ex vivo administration) or target tissues (e.g. invivo administration).

Plasmid delivery involves the cloning of a guide RNA into a CRISPR-Casprotein expressing plasmid and transfecting the DNA in cell culture.Plasmid backbones are available commercially and no specific equipmentis required. They have the advantage of being modular, capable ofcarrying different sizes of CRISPR-Cas coding sequences (including thoseencoding larger sized proteins) as well as selection markers. Both anadvantage of plasmids is that they can ensure transient, but sustainedexpression. However, delivery of plasmids is not straightforward suchthat in vivo efficiency is often low. The sustained expression can alsobe disadvantageous in that it can increase off-target editing. Inaddition excess build-up of the CRISPR-Cas protein can be toxic to thecells. Finally, plasmids always hold the risk of random integration ofthe dsDNA in the host genome, more particularly in view of thedouble-stranded breaks being generated (on and off-target).

The preparation of lipid:nucleic acid complexes, including targetedliposomes such as immunolipid complexes, is well known to one of skillin the art (see, e.g., Crystal, Science 270:404-410 (1995); Blaese etal., Cancer Gene Ther. 2:291-297 (1995); Behr et al., Bioconjugate Chem.5:382-389 (1994); Remy et al., Bioconjugate Chem. 5:647-654 (1994); Gaoet al., Gene Therapy 2:710-722 (1995); Ahmad et al., Cancer Res.52:4817-4820 (1992); U.S. Pat. Nos. 4,186,183, 4,217,344, 4,235,871,4,261,975, 4,485,054, 4,501,728, 4,774,085, 4,837,028, and 4,946,787).This is discussed more in detail below.

The use of RNA or DNA viral based systems for the delivery of nucleicacids takes advantage of highly evolved processes for targeting a virusto specific cells in the body and trafficking the viral payload to thenucleus. Viral vectors can be administered directly to patients (invivo) or they can be used to treat cells in vitro, and the modifiedcells may optionally be administered to patients (ex vivo). Conventionalviral based systems could include retroviral, lentivirus, adenoviral,adeno-associated and herpes simplex virus vectors for gene transfer.Integration in the host genome is possible with the retrovirus,lentivirus, and adeno-associated virus gene transfer methods, oftenresulting in long term expression of the inserted transgene.Additionally, high transduction efficiencies have been observed in manydifferent cell types and target tissues.

The tropism of a retrovirus can be altered by incorporating foreignenvelope proteins, expanding the potential target population of targetcells. Lentiviral vectors are retroviral vectors that are able totransduce or infect non-dividing cells and typically produce high viraltiters. Selection of a retroviral gene transfer system would thereforedepend on the target tissue. Retroviral vectors are comprised ofcis-acting long terminal repeats with packaging capacity for up to 6-10kb of foreign sequence. The minimum cis-acting LTRs are sufficient forreplication and packaging of the vectors, which are then used tointegrate the therapeutic gene into the target cell to provide permanenttransgene expression. Widely used retroviral vectors include those basedupon murine leukemia virus (MuLV), gibbon ape leukemia virus (GaLV),Simian Immuno deficiency virus (SIV), human immuno deficiency virus(HIV), and combinations thereof (see, e.g., Buchscher et al., J. Virol.66:2731-2739 (1992); Johann et al., J. Virol. 66:1635-1640 (1992);Sommnerfelt et al., Virol. 176:58-59 (1990); Wilson et al., J. Virol.63:2374-2378 (1989); Miller et al., J. Virol. 65:2220-2224 (1991);PCT/US94/05700).

In applications where transient expression is preferred, adenoviralbased systems may be used. Adenoviral based vectors are capable of veryhigh transduction efficiency in many cell types and do not require celldivision. With such vectors, high titer and levels of expression havebeen obtained. This vector can be produced in large quantities in arelatively simple system. Adeno-associated virus (“AAV”) vectors mayalso be used to transduce cells with target nucleic acids, e.g., in thein vitro production of nucleic acids and peptides, and for in vivo andex vivo gene therapy procedures (see, e.g., West et al., Virology160:38-47 (1987); U.S. Pat. No. 4,797,368; WO 93/24641; Kotin, HumanGene Therapy 5:793-801 (1994); Muzyczka, J. Clin. Invest. 94:1351(1994). Construction of recombinant AAV vectors are described in anumber of publications, including U.S. Pat. No. 5,173,414; Tratschin etal., Mol. Cell. Biol. 5:3251-3260 (1985); Tratschin, et al., Mol. Cell.Biol. 4:2072-2081 (1984); Hermonat & Muzyczka, PNAS 81:6466-6470 (1984);and Samulski et al., J. Virol. 63:03822-3828 (1989).

The invention provides AAV that contains or consists essentially of anexogenous nucleic acid molecule encoding a CRISPR system, e.g., aplurality of cassettes comprising or consisting a first cassettecomprising or consisting essentially of a promoter, a nucleic acidmolecule encoding a CRISPR-associated (Cas) protein (putative nucleaseor helicase proteins), e.g., Cas13 and a terminator, andone or more,advantageously up to the packaging size limit of the vector, e.g., intotal (including the first cassette) five, cassettes comprising orconsisting essentially of a promoter, nucleic acid molecule encodingguide RNA (gRNA) and a terminator (e.g., each cassette schematicallyrepresented as Promoter-gRNA1-terminator, Promoter-gRNA2-terminator . .. . Promoter-gRNA(N)-terminator, where N is a number that can beinserted that is at an upper limit of the packaging size limit of thevector), or two or more individual rAAVs, each containing one or morethan one cassette of a CRISPR system, e.g., a first rAAV containing thefirst cassette comprising or consisting essentially of a promoter, anucleic acid molecule encoding Cas, e.g., Cas (Cas13) and a terminator,and a second rAAV containing one or more cassettes each comprising orconsisting essentially of a promoter, nucleic acid molecule encodingguide RNA (gRNA) and a terminator (e.g., each cassette schematicallyrepresented as Promoter-gRNA1-terminator, Promoter-gRNA2-terminatorPromoter-gRNA(N)-terminator, where N is a number that can be insertedthat is at an upper limit of the packaging size limit of the vector).Alternatively, because Cas13 can process its own crRNA/gRNA, a singlecrRNA/gRNA array can be used for multiplex gene editing. Hence, insteadof including multiple cassettes to deliver the gRNAs, the rAAV maycontain a single cassette comprising or consisting essentially of apromoter, a plurality of crRNA/gRNA, and a terminator (e.g.,schematically represented as Promoter-gRNA1-gRNA2 . . .gRNA(N)-terminator, where N is a number that can be inserted that is atan upper limit of the packaging size limit of the vector). See Zetscheet al Nature Biotechnology 35, 31-34 (2017), which is incorporatedherein by reference in its entirety. As rAAV is a DNA virus, the nucleicacid molecules in the herein discussion concerning AAV or rAAV areadvantageously DNA. The promoter is in some embodiments advantageouslyhuman Synapsin I promoter (hSyn). Additional methods for the delivery ofnucleic acids to cells are known to those skilled in the art. See, forexample, US20030087817, incorporated herein by reference.

In another embodiment, Cocal vesiculovirus envelope pseudotypedretroviral vector particles are contemplated (see, e.g., US PatentPublication No. 20120164118 assigned to the Fred Hutchinson CancerResearch Center). Cocal virus is in the Vesiculovirus genus, and is acausative agent of vesicular stomatitis in mammals. Cocal virus wasoriginally isolated from mites in Trinidad (Jonkers et al., Am. J. Vet.Res. 25:236-242 (1964)), and infections have been identified inTrinidad, Brazil, and Argentina from insects, cattle, and horses. Manyof the vesiculoviruses that infect mammals have been isolated fromnaturally infected arthropods, suggesting that they are vector-borne.Antibodies to vesiculoviruses are common among people living in ruralareas where the viruses are endemic and laboratory-acquired; infectionsin humans usually result in influenza-like symptoms. The Cocal virusenvelope glycoprotein shares 71.5% identity at the amino acid level withVSV-G Indiana, and phylogenetic comparison of the envelope gene ofvesiculoviruses shows that Cocal virus is serologically distinct from,but most closely related to, VSV-G Indiana strains among thevesiculoviruses. Jonkers et al., Am. J. Vet. Res. 25:236-242 (1964) andTravassos da Rosa et al., Am. J. Tropical Med. & Hygiene 33:999-1006(1984). The Cocal vesiculovirus envelope pseudotyped retroviral vectorparticles may include for example, lentiviral, alpharetroviral,betaretroviral, gammaretroviral, deltaretroviral, and epsilonretroviralvector particles that may comprise retroviral Gag, Pol, and/or one ormore accessory protein(s) and a Cocal vesiculovirus envelope protein.Within certain aspects of these embodiments, the Gag, Pol, and accessoryproteins are lentiviral and/or gammaretroviral.

In some embodiments, a host cell is transiently or non-transientlytransfected with one or more vectors described herein. In someembodiments, a cell is transfected as it naturally occurs in a subjectoptionally to be reintroduced therein. In some embodiments, a cell thatis transfected is taken from a subject. In some embodiments, the cell isderived from cells taken from a subject, such as a cell line. A widevariety of cell lines for tissue culture are known in the art. Examplesof cell lines include, but are not limited to, C8161, CCRF-CEM, MOLT,mIMCD-3, NHDF, HeLa-S3, Huh1, Huh4, Huh7, HUVEC, HASMC, HEKn, HEKa,MiaPaCell, Panc1, PC-3, TF1, CTLL-2, C1R, Rat6, CV1, RPTE, A10, T24,J82, A375, ARH-77, Calu1, SW480, SW620, SKOV3, SK-UT, CaCo2, P388D1,SEM-K2, WEHI-231, HB56, TIB55, Jurkat, J45.01, LRMB, Bcl-1, BC-3, IC21,DLD2, Raw264.7, NRK, NRK-52E, MRC5, MEF, Hep G2, HeLa B, HeLa T4, COS,COS-1, COS-6, COS-M6A, BS-C-1 monkey kidney epithelial, BALB/3T3 mouseembryo fibroblast, 3T3 Swiss, 3T3-L1, 132-d5 human fetal fibroblasts;10.1 mouse fibroblasts, 293-T, 3T3, 721, 9L, A2780, A2780ADR, A2780cis,A172, A20, A253, A431, A-549, ALC, B16, B35, BCP-1 cells, BEAS-2B,bEnd.3, BHK-21, BR 293, BxPC3, C3H-10T1/2, C6/36, Cal-27, CHO, CHO-7,CHO-IR, CHO-K1, CHO-K2, CHO-T, CHO Dhfr−/−, COR-L23, COR-L23/CPR,COR-L23/5010, COR-L23/R23, COS-7, COV-434, CML T1, CMT, CT26, D17, DH82,DU145, DuCaP, EL4, EM2, EM3, EMT6/AR1, EMT6/AR10.0, FM3, H1299, H69,HB54, HB55, HCA2, HEK-293, HeLa, Hepa1c1c7, HL-60, HMEC, HT-29, Jurkat,JY cells, K562 cells, Ku812, KCL22, KG1, KYO1, LNCap, Ma-Mel 1-48,MC-38, MCF-7, MCF-10A, MDA-MB-231, MDA-MB-468, MDA-MB-435, MDCK II, MDCKII, MOR/0.2R, MONO-MAC 6, MTD-1A, MyEnd, NCI-H69/CPR, NCI-H69/LX10,NCI-H69/LX20, NCI-H69/LX4, NIH-3T3, NALM-1, NW-145, OPCN/OPCT celllines, Peer, PNT-1A/PNT 2, RenCa, RIN-5F, RMA/RMAS, Saos-2 cells, Sf-9,SkBr3, T2, T-47D, T84, THP1 cell line, U373, U87, U937, VCaP, Verocells, WM39, WT-49, X63, YAC-1, YAR, and transgenic varieties thereof.Cell lines are available from a variety of sources known to those withskill in the art (see, e.g., the American Type Culture Collection (ATCC)(Manassus, Va.)).

In particular embodiments, transient expression and/or presence of oneor more of the components of the System can be of interest, such as toreduce off-target effects. In some embodiments, a cell transfected withone or more vectors described herein is used to establish a new cellline comprising one or more vector-derived sequences. In someembodiments, a cell transiently transfected with the components of aSystem as described herein (such as by transient transfection of one ormore vectors, or transfection with RNA), and modified through theactivity of a CRISPR complex, is used to establish a new cell linecomprising cells containing the modification but lacking any otherexogenous sequence. In some embodiments, cells transiently ornon-transiently transfected with one or more vectors described herein,or cell lines derived from such cells are used in assessing one or moretest compounds.

In some embodiments it is envisaged to introduce the RNA and/or proteindirectly to the host cell. For instance, the CRISPR-Cas protein can bedelivered as encoding mRNA together with an in vitro transcribed guideRNA. Such methods can reduce the time to ensure effect of the CRISPR-Casprotein and further prevents long-term expression of the CRISPR systemcomponents.

In some embodiments the RNA molecules of the invention are delivered inliposome or lipofectin formulations and the like and can be prepared bymethods well known to those skilled in the art. Such methods aredescribed, for example, in U.S. Pat. Nos. 5,593,972, 5,589,466, and5,580,859, which are herein incorporated by reference. Delivery systemsaimed specifically at the enhanced and improved delivery of siRNA intomammalian cells have been developed, (see, for example, Shen et al FEBSLet. 2003, 539:111-114; Xia et al., Nat. Biotech. 2002, 20:1006-1010;Reich et al., Mol. Vision. 2003, 9: 210-216; Sorensen et al., J. Mol.Biol. 2003, 327: 761-766; Lewis et al., Nat. Gen. 2002, 32: 107-108 andSimeoni et al., NAR 2003, 31, 11: 2717-2724) and may be applied to thepresent invention. siRNA has recently been successfully used forinhibition of gene expression in primates (see for example. Tolentino etal., Retina 24(4):660 which may also be applied to the presentinvention.

Indeed, RNA delivery is a useful method of in vivo delivery. It ispossible to deliver Cas13, adenosine deaminase, and guide RNA into cellsusing liposomes or nanoparticles. Thus delivery of the CRISPR-Casprotein, such as a Cas13, the delivery of the adenosine deaminase (whichmay be fused to the CRISPR-Cas protein or an adaptor protein), and/ordelivery of the RNAs of the invention may be in RNA form and viamicrovesicles, liposomes or particle or particles. For example, Cas13mRNA, adenosine deaminase mRNA, and guide RNA can be packaged intoliposomal particles for delivery in vivo. Liposomal transfectionreagents such as lipofectamine from Life Technologies and other reagentson the market can effectively deliver RNA molecules into the liver.

Means of delivery of RNA also preferred include delivery of RNA viaparticles (Cho, S., Goldberg, M., Son, S., Xu, Q., Yang, F., Mei, Y.,Bogatyrev, S., Langer, R. and Anderson, D., Lipid-like nanoparticles forsmall interfering RNA delivery to endothelial cells, Advanced FunctionalMaterials, 19: 3112-3118, 2010) or exosomes (Schroeder, A., Levins, C.,Cortez, C., Langer, R., and Anderson, D., Lipid-based nanotherapeuticsfor siRNA delivery, Journal of Internal Medicine, 267: 9-21, 2010, PMID:20059641). Indeed, exosomes have been shown to be particularly useful indelivery siRNA, a system with some parallels to the CRISPR system. Forinstance, El-Andaloussi S, et al. (“Exosome-mediated delivery of siRNAin vitro and in vivo.” Nat Protoc. 2012 December; 7(12):2112-26. doi:10.1038/nprot.2012.131. Epub 2012 Nov. 15.) describe how exosomes arepromising tools for drug delivery across different biological barriersand can be harnessed for delivery of siRNA in vitro and in vivo. Theirapproach is to generate targeted exosomes through transfection of anexpression vector, comprising an exosomal protein fused with a peptideligand. The exosomes are then purify and characterized from transfectedcell supernatant, then RNA is loaded into the exosomes. Delivery oradministration according to the invention can be performed withexosomes, in particular but not limited to the brain. Vitamin E(a-tocopherol) may be conjugated with CRISPR Cas and delivered to thebrain along with high density lipoprotein (HDL), for example in asimilar manner as was done by Uno et al. (HUMAN GENE THERAPY 22:711-719(June 2011)) for delivering short-interfering RNA (siRNA) to the brain.Mice were infused via Osmotic minipumps (model 1007D; Alzet, Cupertino,CA) filled with phosphate-buffered saline (PBS) or free TocsiBACE orToc-siBACE/HDL and connected with Brain Infusion Kit 3 (Alzet). Abrain-infusion cannula was placed about 0.5 mm posterior to the bregmaat midline for infusion into the dorsal third ventricle. Uno et al.found that as little as 3 nmol of Toc-siRNA with HDL could induce atarget reduction in comparable degree by the same ICV infusion method. Asimilar dosage of CRISPR Cas conjugated to a-tocopherol andco-administered with HDL targeted to the brain may be contemplated forhumans in the present invention, for example, about 3 nmol to about 3μmol of CRISPR Cas targeted to the brain may be contemplated. Zou et al.((HUMAN GENE THERAPY 22:465-475 (April 2011)) describes a method oflentiviral-mediated delivery of short-hairpin RNAs targeting PKCγ for invivo gene silencing in the spinal cord of rats. Zou et al. administeredabout 10 μl of a recombinant lentivirus having a titer of 1×109transducing units (TU)/ml by an intrathecal catheter. A similar dosageof CRISPR Cas expressed in a lentiviral vector targeted to the brain maybe contemplated for humans in the present invention, for example, about10-50 ml of CRISPR Cas targeted to the brain in a lentivirus having atiter of 1×10⁹ transducing units (TU)/ml may be contemplated.

Dosage of Vectors

In some embodiments, the vector, e.g., plasmid or viral vector isdelivered to the tissue of interest by, for example, an intramuscularinjection, while other times the delivery is via intravenous,transdermal, intranasal, oral, mucosal, or other delivery methods. Suchdelivery may be either via a single dose, or multiple doses. One skilledin the art understands that the actual dosage to be delivered herein mayvary greatly depending upon a variety of factors, such as the vectorchoice, the target cell, organism, or tissue, the general condition ofthe subject to be treated, the degree of transformation/modificationsought, the administration route, the administration mode, the type oftransformation/modification sought, etc.

Such a dosage may further contain, for example, a carrier (water,saline, ethanol, glycerol, lactose, sucrose, calcium phosphate, gelatin,dextran, agar, pectin, peanut oil, sesame oil, etc.), a diluent, apharmaceutically-acceptable carrier (e.g., phosphate-buffered saline), apharmaceutically-acceptable excipient, and/or other compounds known inthe art. The dosage may further contain one or more pharmaceuticallyacceptable salts such as, for example, a mineral acid salt such as ahydrochloride, a hydrobromide, a phosphate, a sulfate, etc.; and thesalts of organic acids such as acetates, propionates, malonates,benzoates, etc. Additionally, auxiliary substances, such as wetting oremulsifying agents, pH buffering substances, gels or gelling materials,flavorings, colorants, microspheres, polymers, suspension agents, etc.may also be present herein. In addition, one or more other conventionalpharmaceutical ingredients, such as preservatives, humectants,suspending agents, surfactants, antioxidants, anticaking agents,fillers, chelating agents, coating agents, chemical stabilizers, etc.may also be present, especially if the dosage form is a reconstitutableform. Suitable exemplary ingredients include microcrystalline cellulose,carboxymethylcellulose sodium, polysorbate 80, phenylethyl alcohol,chlorobutanol, potassium sorbate, sorbic acid, sulfur dioxide, propylgallate, the parabens, ethyl vanillin, glycerin, phenol,parachlorophenol, gelatin, albumin and a combination thereof. A thoroughdiscussion of pharmaceutically acceptable excipients is available inREMINGTON′S PHARMACEUTICAL SCIENCES (Mack Pub. Co., N.J. 1991) which isincorporated by reference herein.

In an embodiment herein the delivery is via an adenovirus, which may beat a single booster dose containing at least 1×105 particles (alsoreferred to as particle units, pu) of adenoviral vector. In anembodiment herein, the dose preferably is at least about 1×106 particles(for example, about 1×106-1×1012 particles), more preferably at leastabout 1×107 particles, more preferably at least about 1×108 particles(e.g., about 1×108-1×1011 particles or about 1×108-1×1012 particles),and most preferably at least about 1×100 particles (e.g., about1×109-1×1010 particles or about 1×109-1×1012 particles), or even atleast about 1×1010 particles (e.g., about 1×1010-1×1012 particles) ofthe adenoviral vector. Alternatively, the dose comprises no more thanabout 1×1014 particles, preferably no more than about 1×1013 particles,even more preferably no more than about 1×1012 particles, even morepreferably no more than about 1×1011 particles, and most preferably nomore than about 1×1010 particles (e.g., no more than about 1×109articles). Thus, the dose may contain a single dose of adenoviral vectorwith, for example, about 1×106 particle units (pu), about 2×106 pu,about 4×106 pu, about 1×107 pu, about 2×107 pu, about 4×107 pu, about1×108 pu, about 2×108 pu, about 4×108 pu, about 1×109 pu, about 2×109pu, about 4×109 pu, about 1×1010 pu, about 2×1010 pu, about 4×1010 pu,about 1×1011 pu, about 2×1011 pu, about 4×1011 pu, about 1×1012 pu,about 2×1012 pu, or about 4×1012 pu of adenoviral vector. See, forexample, the adenoviral vectors in U.S. Pat. No. 8,454,972 B2 to Nabel,et. al., granted on June 4, 2013; incorporated by reference herein, andthe dosages at col 29, lines 36-58 thereof. In an embodiment herein, theadenovirus is delivered via multiple doses.

In an embodiment herein, the delivery is via an AAV. A therapeuticallyeffective dosage for in vivo delivery of the AAV to a human is believedto be in the range of from about 20 to about 50 ml of saline solutioncontaining from about 1×1010 to about 1×1010 functional AAV/ml solution.The dosage may be adjusted to balance the therapeutic benefit againstany side effects. In an embodiment herein, the AAV dose is generally inthe range of concentrations of from about 1×105 to 1×1050 genomes AAV,from about 1×108 to 1×1020 genomes AAV, from about 1×1010 to about1×1016 genomes, or about 1×1011 to about 1×1016 genomes AAV. A humandosage may be about 1×1013 genomes AAV. Such concentrations may bedelivered in from about 0.001 ml to about 100 ml, about 0.05 to about 50ml, or about 10 to about 25 ml of a carrier solution. Other effectivedosages can be readily established by one of ordinary skill in the artthrough routine trials establishing dose response curves. See, forexample, U.S. Pat. No. 8,404,658 B2 to Hajjar, et al., granted on Mar.26, 2013, at col. 27, lines 45-60.

In an embodiment herein the delivery is via a plasmid. In such plasmidcompositions, the dosage should be a sufficient amount of plasmid toelicit a response. For instance, suitable quantities of plasmid DNA inplasmid compositions can be from about 0.1 to about 2 mg, or from about1 μg to about 10 μg per 70 kg individual. Plasmids of the invention willgenerally comprise (i) a promoter; (ii) a sequence encoding a CRISPR-Casprotein, operably linked to said promoter; (iii) a selectable marker;(iv) an origin of replication; and (v) a transcription terminatordownstream of and operably linked to (ii). The plasmid can also encodethe RNA components of a CRISPR complex, but one or more of these mayinstead be encoded on a different vector.

The doses herein are based on an average 70 kg individual. The frequencyof administration is within the ambit of the medical or veterinarypractitioner (e.g., physician, veterinarian), or scientist skilled inthe art. It is also noted that mice used in experiments are typicallyabout 20 g and from mice experiments one can scale up to a 70 kgindividual.

The dosage used for the compositions provided herein include dosages forrepeated administration or repeat dosing. In particular embodiments, theadministration is repeated within a period of several weeks, months, oryears. Suitable assays can be performed to obtain an optimal dosageregime. Repeated administration can allow the use of lower dosage, whichcan positively affect off-target modifications.

RNA Delivery

In particular embodiments, RNA based delivery is used. In theseembodiments, mRNA of the CRISPR-Cas protein, mRNA of the adenosinedeaminase (which may be fused to a CRISPR-Cas protein or an adaptor),are delivered together with in vitro transcribed guide RNA. Liang et al.describes efficient genome editing using RNA based delivery (ProteinCell. 2015 May; 6(5): 363-372). In some embodiments, the mRNA(s)encoding Cas13 and/or adenosine deaminase can be chemically modified,which may lead to improved activity compared to plasmid-encoded Cas13and/or adenosine deaminase. For example, uridines in the mRNA(s) can bepartially or fully substituted with pseudouridine (Ψ),N1-methylpseudouridine (me1Ψ), 5-methoxyuridine(5moU). See Li et al.,Nature Biomedical Engineering 1, 0066 D01:10.1038/s41551-017-0066(2017), which is incorporated herein by reference in its entirety.

RNP Delivery

In particular embodiments, pre-complexed guide RNA, CRISPR-Cas protein,and adenosine deaminase (which may be fused to a CRISPR-Cas protein oran adaptor) are delivered as a ribonucleoprotein (RNP). RNPs have theadvantage that they lead to rapid editing effects even more so than theRNA method because this process avoids the need for transcription. Animportant advantage is that both RNP delivery is transient, reducingoff-target effects and toxicity issues. Efficient genome editing indifferent cell types has been observed by Kim et al. (2014, Genome Res.24(6):1012-9), Paix et al. (2015, Genetics 204(1):47-54), Chu et al.(2016, BMC Biotechnol. 16:4), and Wang et al. (2013, Cell. 9;153(4):910-8).

In particular embodiments, the ribonucleoprotein is delivered by way ofa polypeptide-based shuttle agent as described in WO2016161516.WO2016161516 describes efficient transduction of polypeptide cargosusing synthetic peptides comprising an endosome leakage domain (ELD)operably linked to a cell penetrating domain (CPD), to a histidine-richdomain and a CPD. Similarly these polypeptides can be used for thedelivery of CRISPR-effector based RNPs in eukaryotic cells.

Particles

In some aspects or embodiments, a composition comprising a deliveryparticle formulation may be used. In some aspects or embodiments, theformulation comprises a CRISPR complex, the complex comprising a CRISPRprotein and a guide which directs sequence-specific binding of theCRISPR complex to a target sequence. In some embodiments, the deliveryparticle comprises a lipid-based particle, optionally a lipidnanoparticle, or cationic lipid and optionally biodegradable polymer. Insome embodiments, the cationic lipid comprises1,2-dioleoyl-3-trimethylammonium-propane (DOTAP). In some embodiments,the hydrophilic polymer comprises ethylene glycol or polyethyleneglycol. In some embodiments, the delivery particle further comprises alipoprotein, preferably cholesterol. In some embodiments, the deliveryparticles are less than 500 nm in diameter, optionally less than 250 nmin diameter, optionally less than 100 nm in diameter, optionally about35 nm to about 60 nm in diameter.

Example particle delivery complexes are further disclosed in U.S.Provisional Application entitled “Nove Delivery of Large Payloads” filed62/485,625 filed Apr. 14, 2017.

Several types of particle delivery systems and/or formulations are knownto be useful in a diverse spectrum of biomedical applications. Ingeneral, a particle is defined as a small object that behaves as a wholeunit with respect to its transport and properties. Particles are furtherclassified according to diameter. Coarse particles cover a range between2,500 and 10,000 nanometers. Fine particles are sized between 100 and2,500 nanometers. Ultrafine particles, or nanoparticles, are generallybetween 1 and 100 nanometers in size. The basis of the 100-nm limit isthe fact that novel properties that differentiate particles from thebulk material typically develop at a critical length scale of under 100nm.

As used herein, a particle delivery system/formulation is defined as anybiological delivery system/formulation which includes a particle inaccordance with the present invention. A particle in accordance with thepresent invention is any entity having a greatest dimension (e.g.diameter) of less than 100 microns (μm). In some embodiments, inventiveparticles have a greatest dimension of less than 10 μm. In someembodiments, inventive particles have a greatest dimension of less than2000 nanometers (nm). In some embodiments, inventive particles have agreatest dimension of less than 1000 nanometers (nm). In someembodiments, inventive particles have a greatest dimension of less than900 nm, 800 nm, 700 nm, 600 nm, 500 nm, 400 nm, 300 nm, 200 nm, or 100nm. Typically, inventive particles have a greatest dimension (e.g.,diameter) of 500 nm or less. In some embodiments, inventive particleshave a greatest dimension (e.g., diameter) of 250 nm or less. In someembodiments, inventive particles have a greatest dimension (e.g.,diameter) of 200 nm or less. In some embodiments, inventive particleshave a greatest dimension (e.g., diameter) of 150 nm or less. In someembodiments, inventive particles have a greatest dimension (e.g.,diameter) of 100 nm or less. Smaller particles, e.g., having a greatestdimension of 50 nm or less are used in some embodiments of theinvention. In some embodiments, inventive particles have a greatestdimension ranging between 25 nm and 200 nm.

In terms of this invention, it is preferred to have one or morecomponents of CRISPR complex, e.g., CRISPR-Cas protein or mRNA, oradenosine deaminase (which may be fused to a CRISPR-Cas protein or anadaptor) or mRNA, or guide RNA delivered using nanoparticles or lipidenvelopes. Other delivery systems or vectors are may be used inconjunction with the nanoparticle aspects of the invention.

In general, a “nanoparticle” refers to any particle having a diameter ofless than 1000 nm. In certain preferred embodiments, nanoparticles ofthe invention have a greatest dimension (e.g., diameter) of 500 nm orless. In other preferred embodiments, nanoparticles of the inventionhave a greatest dimension ranging between 25 nm and 200 nm. In otherpreferred embodiments, nanoparticles of the invention have a greatestdimension of 100 nm or less. In other preferred embodiments,nanoparticles of the invention have a greatest dimension ranging between35 nm and 60 nm. It will be appreciated that reference made herein toparticles or nanoparticles can be interchangeable, where appropriate.

It will be understood that the size of the particle will differdepending as to whether it is measured before or after loading.Accordingly, in particular embodiments, the term “nanoparticles” mayapply only to the particles pre loading.

Nanoparticles encompassed in the present invention may be provided indifferent forms, e.g., as solid nanoparticles (e.g., metal such assilver, gold, iron, titanium), non-metal, lipid-based solids, polymers),suspensions of nanoparticles, or combinations thereof. Metal,dielectric, and semiconductor nanoparticles may be prepared, as well ashybrid structures (e.g., core-shell nanoparticles). Nanoparticles madeof semiconducting material may also be labeled quantum dots if they aresmall enough (typically sub 10 nm) that quantization of electronicenergy levels occurs. Such nanoscale particles are used in biomedicalapplications as drug carriers or imaging agents and may be adapted forsimilar purposes in the present invention.

Semi-solid and soft nanoparticles have been manufactured, and are withinthe scope of the present invention. A prototype nanoparticle ofsemi-solid nature is the liposome. Various types of liposomenanoparticles are currently used clinically as delivery systems foranticancer drugs and vaccines. Nanoparticles with one half hydrophilicand the other half hydrophobic are termed Janus particles and areparticularly effective for stabilizing emulsions. They can self-assembleat water/oil interfaces and act as solid surfactants.

Particle characterization (including e.g., characterizing morphology,dimension, etc.) is done using a variety of different techniques. Commontechniques are electron microscopy (TEM, SEM), atomic force microscopy(AFM), dynamic light scattering (DLS), X-ray photoelectron spectroscopy(XPS), powder X-ray diffraction (XRD), Fourier transform infraredspectroscopy (FTIR), matrix-assisted laser desorption/ionizationtime-of-flight mass spectrometry(MALDI-TOF), ultraviolet-visiblespectroscopy, dual polarization interferometry and nuclear magneticresonance (NMR). Characterization (dimension measurements) may be madeas to native particles (i.e., preloading) or after loading of the cargo(herein cargo refers to e.g., one or more components of CRISPR-Cassystem e.g., CRISPR-Cas protein or mRNA, adenosine deaminase (which maybe fused to a CRISPR-Cas protein or an adaptor) or mRNA, or guide RNA,or any combination thereof, and may include additional carriers and/orexcipients) to provide particles of an optimal size for delivery for anyin vitro, ex vivo and/or in vivo application of the present invention.In certain preferred embodiments, particle dimension (e.g., diameter)characterization is based on measurements using dynamic laser scattering(DLS). Mention is made of U.S. Pat. Nos. 8,709,843; 6,007,845;5,855,913; 5,985,309; 5,543,158; and the publication by James E. Dahlmanand Carmen Barnes et al. Nature Nanotechnology (2014) published online11 May 2014, doi:10.1038/nnano.2014.84, concerning particles, methods ofmaking and using them and measurements thereof.

Particles delivery systems within the scope of the present invention maybe provided in any form, including but not limited to solid, semi-solid,emulsion, or colloidal particles. As such any of the delivery systemsdescribed herein, including but not limited to, e.g., lipid-basedsystems, liposomes, micelles, microvesicles, exosomes, or gene gun maybe provided as particle delivery systems within the scope of the presentinvention.

CRISPR-Cas protein mRNA, adenosine deaminase (which may be fused to aCRISPR-Cas protein or an adaptor) or mRNA, and guide RNA may bedelivered simultaneously using particles or lipid envelopes; forinstance, CRISPR-Cas protein and RNA of the invention, e.g., as acomplex, can be delivered via a particle as in Dahlman et al.,WO2015089419 A2 and documents cited therein, such as 7C1 (see, e.g.,James E. Dahlman and Carmen Barnes et al. Nature Nanotechnology (2014)published online 11 May 2014, doi:10.1038/nnano.2014.84), e.g., deliveryparticle comprising lipid or lipidoid and hydrophilic polymer, e.g.,cationic lipid and hydrophilic polymer, for instance wherein thecationic lipid comprises 1,2-dioleoyl-3-trimethylammonium-propane(DOTAP) or 1,2-ditetradecanoyl-sn-glycero-3-phosphocholine (DMPC) and/orwherein the hydrophilic polymer comprises ethylene glycol orpolyethylene glycol (PEG); and/or wherein the particle further comprisescholesterol (e.g., particle from formulation 1=DOTAP 100, DMPC 0, PEG 0,Cholesterol 0; formulation number 2=DOTAP 90, DMPC 0, PEG 10,Cholesterol 0; formulation number 3=DOTAP 90, DMPC 0, PEG 5, Cholesterol5), wherein particles are formed using an efficient, multistep processwherein first, effector protein and RNA are mixed together, e.g., at a1:1 molar ratio, e.g., at room temperature, e.g., for 30 minutes, e.g.,in sterile, nuclease free 1× PBS; and separately, DOTAP, DMPC, PEG, andcholesterol as applicable for the formulation are dissolved in alcohol,e.g., 100% ethanol; and, the two solutions are mixed together to formparticles containing the complexes).

Nucleic acid-targeting effector proteins (e.g., a Type V protein such asCas13) mRNA and guide RNA may be delivered simultaneously usingparticles or lipid envelopes. Examples of suitable particles include butare not limited to those described in U.S. Pat. No. 9,301,923.

For example, Su X, Fricke J, Kavanagh D G, Irvine D J (“In vitro and invivo mRNA delivery using lipid-enveloped pH-responsive polymernanoparticles” Mol Pharm. 2011 Jun. 6; 8(3):774-87. doi:10.1021/mp100390w. Epub 2011 Apr. 1) describes biodegradable core-shellstructured nanoparticles with a poly(β-amino ester) (PBAE) coreenveloped by a phospholipid bilayer shell. These were developed for invivo mRNA delivery. The pH-responsive PBAE component was chosen topromote endosome disruption, while the lipid surface layer was selectedto minimize toxicity of the polycation core. Such are, therefore,preferred for delivering RNA of the present invention.

In one embodiment, particles/nanoparticles based on self assemblingbioadhesive polymers are contemplated, which may be applied to oraldelivery of peptides, intravenous delivery of peptides and nasaldelivery of peptides, all to the brain. Other embodiments, such as oralabsorption and ocular delivery of hydrophobic drugs are alsocontemplated. The molecular envelope technology involves an engineeredpolymer envelope which is protected and delivered to the site of thedisease (see, e.g., Mazza, M. et al. ACSNano, 2013. 7(2): 1016-1026;Siew, A., et al. Mol Pharm, 2012. 9(1):14-28; Lalatsa, A., et al. JContr Rel, 2012. 161(2):523-36; Lalatsa, A., et al., Mol Pharm, 2012.9(6):1665-80; Lalatsa, A., et al. Mol Pharm, 2012. 9(6):1764-74;Garrett, N. L., et al. J Biophotonics, 2012. 5(5-6):458-68; Garrett, N.L., et al. J Raman Spect, 2012. 43(5):681-688; Ahmad, S., et al. J RoyalSoc Interface 2010. 7:S423-33; Uchegbu, I. F. Expert Opin Drug Deliv,2006. 3(5):629-40; Qu, X., et al. Biomacromolecules, 2006. 7(12):3452-9and Uchegbu, I. F., et al. Int J Pharm, 2001. 224:185-199). Doses ofabout 5 mg/kg are contemplated, with single or multiple doses, dependingon the target tissue.

In one embodiment, particles/nanoparticles that can deliver RNA to acancer cell to stop tumor growth developed by Dan Anderson's lab at MITmay be used/and or adapted to the AD-functionalized CRISPR-Cas system ofthe present invention. In particular, the Anderson lab developed fullyautomated, combinatorial systems for the synthesis, purification,characterization, and formulation of new biomaterials andnanoformulations. See, e.g., Alabi et al., Proc Natl Acad Sci U S A.2013 Aug. 6; 110(32):12881-6; Zhang et al., Adv Mater. 2013 Sep. 6;25(33):4641-5; Jiang et al., Nano Lett. 2013 Mar. 13; 13(3):1059-64;Karagiannis et al., ACS Nano. 2012 Oct. 23; 6(10):8484-7; Whitehead etal., ACS Nano. 2012 Aug. 28; 6(8):6922-9 and Lee et al., NatNanotechnol. 2012 Jun. 3; 7(6):389-93.

US patent application 20110293703 relates to lipidoid compounds are alsoparticularly useful in the administration of polynucleotides, which maybe applied to deliver the AD-functionalized CRISPR-Cas system of thepresent invention. In one aspect, the aminoalcohol lipidoid compoundsare combined with an agent to be delivered to a cell or a subject toform microparticles, nanoparticles, liposomes, or micelles. The agent tobe delivered by the particles, liposomes, or micelles may be in the formof a gas, liquid, or solid, and the agent may be a polynucleotide,protein, peptide, or small molecule. The aminoalcohol lipidoid compoundsmay be combined with other aminoalcohol lipidoid compounds, polymers(synthetic or natural), surfactants, cholesterol, carbohydrates,proteins, lipids, etc. to form the particles. These particles may thenoptionally be combined with a pharmaceutical excipient to form apharmaceutical composition.

US Patent Publication No. 20110293703 also provides methods of preparingthe aminoalcohol lipidoid compounds. One or more equivalents of an amineare allowed to react with one or more equivalents of anepoxide-terminated compound under suitable conditions to form anaminoalcohol lipidoid compound of the present invention. In certainembodiments, all the amino groups of the amine are fully reacted withthe epoxide-terminated compound to form tertiary amines. In otherembodiments, all the amino groups of the amine are not fully reactedwith the epoxide-terminated compound to form tertiary amines therebyresulting in primary or secondary amines in the aminoalcohol lipidoidcompound. These primary or secondary amines are left as is or may bereacted with another electrophile such as a different epoxide-terminatedcompound. As will be appreciated by one skilled in the art, reacting anamine with less than excess of epoxide-terminated compound will resultin a plurality of different aminoalcohol lipidoid compounds with variousnumbers of tails. Certain amines may be fully functionalized with twoepoxide-derived compound tails while other molecules will not becompletely functionalized with epoxide-derived compound tails. Forexample, a diamine or polyamine may include one, two, three, or fourepoxide-derived compound tails off the various amino moieties of themolecule resulting in primary, secondary, and tertiary amines. Incertain embodiments, all the amino groups are not fully functionalized.In certain embodiments, two of the same types of epoxide-terminatedcompounds are used. In other embodiments, two or more differentepoxide-terminated compounds are used. The synthesis of the aminoalcohollipidoid compounds is performed with or without solvent, and thesynthesis may be performed at higher temperatures ranging from 30-100°C., preferably at approximately 50-90° C. The prepared aminoalcohollipidoid compounds may be optionally purified. For example, the mixtureof aminoalcohol lipidoid compounds may be purified to yield anaminoalcohol lipidoid compound with a particular number ofepoxide-derived compound tails. Or the mixture may be purified to yielda particular stereo- or regioisomer. The aminoalcohol lipidoid compoundsmay also be alkylated using an alkyl halide (e.g., methyl iodide) orother alkylating agent, and/or they may be acylated.

US Patent Publication No. 20110293703 also provides libraries ofaminoalcohol lipidoid compounds prepared by the inventive methods. Theseaminoalcohol lipidoid compounds may be prepared and/or screened usinghigh-throughput techniques involving liquid handlers, robots, microtiterplates, computers, etc. In certain embodiments, the aminoalcohollipidoid compounds are screened for their ability to transfectpolynucleotides or other agents (e.g., proteins, peptides, smallmolecules) into the cell.

US Patent Publication No. 20130302401 relates to a class ofpoly(beta-amino alcohols) (PBAAs) has been prepared using combinatorialpolymerization. The inventive PBAAs may be used in biotechnology andbiomedical applications as coatings (such as coatings of films ormultilayer films for medical devices or implants), additives, materials,excipients, non-biofouling agents, micropatterning agents, and cellularencapsulation agents. When used as surface coatings, these PBAAselicited different levels of inflammation, both in vitro and in vivo,depending on their chemical structures. The large chemical diversity ofthis class of materials allowed us to identify polymer coatings thatinhibit macrophage activation in vitro. Furthermore, these coatingsreduce the recruitment of inflammatory cells, and reduce fibrosis,following the subcutaneous implantation of carboxylated polystyrenemicroparticles. These polymers may be used to form polyelectrolytecomplex capsules for cell encapsulation. The invention may also havemany other biological applications such as antimicrobial coatings, DNAor siRNA delivery, and stem cell tissue engineering. The teachings of USPatent Publication No. 20130302401 may be applied to theAD-functionalized CRISPR-Cas system of the present invention.

Preassembled recombinant CRISPR-Cas complexes comprising Cas13,adenosine deaminase (which may be fused to Cas13 or an adaptor protein),and guide RNA may be transfected, for example by electroporation,resulting in high mutation rates and absence of detectable off-targetmutations. Hur, J. K. et al, Targeted mutagenesis in mice byelectroporation of Cas13 ribonucleoproteins, Nat Biotechnol. 2016 Jun.6. doi: 10.1038/nbt.3596.

In terms of local delivery to the brain, this can be achieved in variousways. For instance, material can be delivered intrastriatally e.g. byinjection. Injection can be performed stereotactically via a craniotomy.

In some embodiments, sugar-based particles may be used, for exampleGalNAc, as described herein and with reference to WO2014118272(incorporated herein by reference) and Nair, J K et al., 2014, Journalof the American Chemical Society 136 (49), 16958-16961) and the teachingherein, especially in respect of delivery applies to all particlesunless otherwise apparent. This may be considered to be a sugar-basedparticle and further details on other particle delivery systems and/orformulations are provided herein. GalNAc can therefore be considered tobe a particle in the sense of the other particles described herein, suchthat general uses and other considerations, for instance delivery ofsaid particles, apply to GalNAc particles as well. A solution-phaseconjugation strategy may for example be used to attach triantennaryGalNAc clusters (mol. wt. ˜2000) activated as PFP (pentafluorophenyl)esters onto 5′-hexylamino modified oligonucleotides (5′-HA ASOs, mol.wt. ˜8000 Da; Østergaard et al., Bioconjugate Chem., 2015, 26 (8), pp1451-1455). Similarly, poly(acrylate) polymers have been described forin vivo nucleic acid delivery (see WO2013158141 incorporated herein byreference). In further alternative embodiments, pre-mixing CRISPRnanoparticles (or protein complexes) with naturally occurring serumproteins may be used in order to improve delivery (Akinc A et al, 2010,Molecular Therapy vol. 18 no. 7, 1357-1364).

Nanoclews

Further, the System may be delivered using nanoclews, for example asdescribed in Sun W et al, Cocoon-like self-degradable DNA nanoclew foranticancer drug delivery., J Am Chem Soc. 2014 Oct. 22; 136(42):14722-5.doi: 10.1021/ja5088024. Epub 2014 Oct. 13.; or in Sun W et al,Self-Assembled DNA Nanoclews for the Efficient Delivery of CRISPR-Cas9for Genome Editing., Angew Chem Int Ed Engl. 2015 Oct. 5;54(41):12029-33. doi: 10.1002/anie.201506030. Epub 2015 Aug. 27.

LNP

In some embodiments, delivery is by encapsulation of the Cas13 proteinor mRNA form in a lipid particle such as an LNP. In some embodiments,therefore, lipid nanoparticles (LNPs) are contemplated. Anantitransthyretin small interfering RNA has been encapsulated in lipidnanoparticles and delivered to humans (see, e.g., Coelho et al., N EnglJ Med 2013; 369:819-29), and such a system may be adapted and applied tothe CRISPR Cas system of the present invention. Doses of about 0.01 toabout 1 mg per kg of body weight administered intravenously arecontemplated. Medications to reduce the risk of infusion-relatedreactions are contemplated, such as dexamethasone, acetampinophen,diphenhydramine or cetirizine, and ranitidine are contemplated. Multipledoses of about 0.3 mg per kilogram every 4 weeks for five doses are alsocontemplated.

LNPs have been shown to be highly effective in delivering siRNAs to theliver (see, e.g., Tabernero et al., Cancer Discovery, April 2013, Vol.3, No. 4, pages 363-470) and are therefore contemplated for deliveringRNA encoding CRISPR Cas to the liver. A dosage of about four doses of 6mg/kg of the LNP every two weeks may be contemplated. Tabernero et al.demonstrated that tumor regression was observed after the first 2 cyclesof LNPs dosed at 0.7 mg/kg, and by the end of 6 cycles the patient hadachieved a partial response with complete regression of the lymph nodemetastasis and substantial shrinkage of the liver tumors. A completeresponse was obtained after 40 doses in this patient, who has remainedin remission and completed treatment after receiving doses over 26months. Two patients with RCC and extrahepatic sites of diseaseincluding kidney, lung, and lymph nodes that were progressing followingprior therapy with VEGF pathway inhibitors had stable disease at allsites for approximately 8 to 12 months, and a patient with PNET andliver metastases continued on the extension study for 18 months (36doses) with stable disease.

However, the charge of the LNP must be taken into consideration. Ascationic lipids combined with negatively charged lipids to inducenonbilayer structures that facilitate intracellular delivery. Becausecharged LNPs are rapidly cleared from circulation following intravenousinjection, ionizable cationic lipids with pKa values below 7 weredeveloped (see, e.g., Rosin et al, Molecular Therapy, vol. 19, no. 12,pages 1286-2200, December 2011). Negatively charged polymers such as RNAmay be loaded into LNPs at low pH values (e.g., pH 4) where theionizable lipids display a positive charge. However, at physiological pHvalues, the LNPs exhibit a low surface charge compatible with longercirculation times. Four species of ionizable cationic lipids have beenfocused upon, namely 1,2-dilineoyl-3-dimethylammonium-propane (DLinDAP),1,2-dilinoleyloxy-3-N,N-dimethylaminopropane (DLinDMA),1,2-dilinoleyloxy-keto-N,N-dimethyl-3-aminopropane (DLinKDMA), and1,2-dilinoleyl-4-(2-dimethylaminoethyl)-[1,3]-dioxolane (DLinKC2-DMA).It has been shown that LNP siRNA systems containing these lipids exhibitremarkably different gene silencing properties in hepatocytes in vivo,with potencies varying according to the seriesDLinKC2-DMA>DLinKDMA>DLinDMA>>DLinDAP employing a Factor VII genesilencing model (see, e.g., Rosin et al, Molecular Therapy, vol. 19, no.12, pages 1286-2200, December 2011). A dosage of 1 μg/ml of LNP orCRISPR-Cas RNA in or associated with the LNP may be contemplated,especially for a formulation containing DLinKC2-DMA.

Preparation of LNPs and CRISPR Cas encapsulation may be used/and oradapted from Rosin et al, Molecular Therapy, vol. 19, no. 12, pages1286-2200, December 2011). The cationic lipids1,2-dilineoyl-3-dimethylammonium-propane (DLinDAP),1,2-dilinoleyloxy-3-N,N-dimethylaminopropane (DLinDMA),1,2-dilinoleyloxyketo-N,N-dimethyl-3-aminopropane (DLinK-DMA),1,2-dilinoleyl-4-(2-dimethylaminoethyl)-[1,3]-dioxolane (DLinKC2-DMA),(3-o-[2″-(methoxypolyethyleneglycol 2000)succinoyl]-1,2-dimyristoyl-sn-glycol (PEG-S-DMG), andR-3-[(ω-methoxy-poly(ethylene glycol)2000)carbamoyl]-1,2-dimyristyloxlpropyl-3-amine (PEG-C-DOMG) may be providedby Tekmira Pharmaceuticals (Vancouver, Canada) or synthesized.Cholesterol may be purchased from Sigma (St Louis, Mo.). The specificCRISPR Cas RNA may be encapsulated in LNPs containing DLinDAP, DLinDMA,DLinK-DMA, and DLinKC2-DMA (cationic lipid:DSPC:CHOL: PEGS-DMG orPEG-C-DOMG at 40:10:40:10 molar ratios). When required, 0.2% SP-DiOC18(Invitrogen, Burlington, Canada) may be incorporated to assess cellularuptake, intracellular delivery, and biodistribution. Encapsulation maybe performed by dissolving lipid mixtures comprised of cationiclipid:DSPC:cholesterol:PEG-c-DOMG (40:10:40:10 molar ratio) in ethanolto a final lipid concentration of 10 mmol/l. This ethanol solution oflipid may be added drop-wise to 50 mmol/l citrate, pH 4.0 to formmultilamellar vesicles to produce a final concentration of 30% ethanolvol/vol. Large unilamellar vesicles may be formed following extrusion ofmultilamellar vesicles through two stacked 80 nm Nuclepore polycarbonatefilters using the Extruder (Northern Lipids, Vancouver, Canada).Encapsulation may be achieved by adding RNA dissolved at 2 mg/ml in 50mmol/l citrate, pH 4.0 containing 30% ethanol vol/vol drop-wise toextruded preformed large unilamellar vesicles and incubation at 31° C.for 30 minutes with constant mixing to a final RNA/lipid weight ratio of0.06/1 wt/wt. Removal of ethanol and neutralization of formulationbuffer were performed by dialysis against phosphate-buffered saline(PBS), pH 7.4 for 16 hours using Spectra/Por 2 regenerated cellulosedialysis membranes. Nanoparticle size distribution may be determined bydynamic light scattering using a NICOMP 370 particle sizer, thevesicle/intensity modes, and Gaussian fitting (Nicomp Particle Sizing,Santa Barbara, Calif.). The particle size for all three LNP systems maybe ˜70 nm in diameter. RNA encapsulation efficiency may be determined byremoval of free RNA using VivaPureD MiniH columns (Sartorius StedimBiotech) from samples collected before and after dialysis. Theencapsulated RNA may be extracted from the eluted nanoparticles andquantified at 260 nm. RNA to lipid ratio was determined by measurementof cholesterol content in vesicles using the Cholesterol E enzymaticassay from Wako Chemicals USA (Richmond, VA). In conjunction with theherein discussion of LNPs and PEG lipids, PEGylated liposomes or LNPsare likewise suitable for delivery of a CRISPR-Cas system or componentsthereof.

A lipid premix solution (20.4 mg/ml total lipid concentration) may beprepared in ethanol containing DLinKC2-DMA, DSPC, and cholesterol at50:10:38.5 molar ratios. Sodium acetate may be added to the lipid premixat a molar ratio of 0.75:1 (sodium acetate:DLinKC2-DMA). The lipids maybe subsequently hydrated by combining the mixture with 1.85 volumes ofcitrate buffer (10 mmol/l, pH 3.0) with vigorous stirring, resulting inspontaneous liposome formation in aqueous buffer containing 35% ethanol.The liposome solution may be incubated at 37° C. to allow fortime-dependent increase in particle size. Aliquots may be removed atvarious times during incubation to investigate changes in liposome sizeby dynamic light scattering (Zetasizer Nano ZS, Malvern Instruments,Worcestershire, UK). Once the desired particle size is achieved, anaqueous PEG lipid solution (stock=10 mg/ml PEG-DMG in 35% (vol/vol)ethanol) may be added to the liposome mixture to yield a final PEG molarconcentration of 3.5% of total lipid. Upon addition of PEG-lipids, theliposomes should their size, effectively quenching further growth. RNAmay then be added to the empty liposomes at an RNA to total lipid ratioof approximately 1:10 (wt:wt), followed by incubation for 30 minutes at37° C. to form loaded LNPs. The mixture may be subsequently dialyzedovernight in PBS and filtered with a 0.45-μm syringe filter.

Spherical Nucleic Acid (SNA™) constructs and other nanoparticles(particularly gold nanoparticles) are also contemplated as a means todelivery CRISPR-Cas system to intended targets. Significant data showthat AuraSense Therapeutics' Spherical Nucleic Acid (SNA™) constructs,based upon nucleic acid-functionalized gold nanoparticles, are useful.

Literature that may be employed in conjunction with herein teachingsinclude: Cutler et al., J. Am. Chem. Soc. 2011 133:9254-9257, Hao etal., Small. 2011 7:3158-3162, Zhang et al., ACS Nano. 2011 5:6962-6970,Cutler et al., J. Am. Chem. Soc. 2012 134:1376-1391, Young et al., NanoLett. 2012 12:3867-71, Zheng et al., Proc. Natl. Acad. Sci. USA. 2012109:11975-80, Mirkin, Nanomedicine 2012 7:635-638 Zhang et al., J. Am.Chem. Soc. 2012 134:16488-1691, Weintraub, Nature 2013 495:S14-S16, Choiet al., Proc. Natl. Acad. Sci. USA. 2013 110(19):7625-7630, Jensen etal., Sci. Transl. Med. 5, 209ra152 (2013) and Mirkin, et al., Small,10:186-192.

Self-assembling nanoparticles with RNA may be constructed withpolyethyleneimine (PEI) that is PEGylated with an Arg-Gly-Asp (RGD)peptide ligand attached at the distal end of the polyethylene glycol(PEG). This system has been used, for example, as a means to targettumor neovasculature expressing integrins and deliver siRNA inhibitingvascular endothelial growth factor receptor-2 (VEGF R2) expression andthereby achieve tumor angiogenesis (see, e.g., Schiffelers et al.,Nucleic Acids Research, 2004, Vol. 32, No. 19). Nanoplexes may beprepared by mixing equal volumes of aqueous solutions of cationicpolymer and nucleic acid to give a net molar excess of ionizablenitrogen (polymer) to phosphate (nucleic acid) over the range of 2 to 6.The electrostatic interactions between cationic polymers and nucleicacid resulted in the formation of polyplexes with average particle sizedistribution of about 100 nm, hence referred to here as nanoplexes. Adosage of about 100 to 200 mg of CRISPR Cas is envisioned for deliveryin the self-assembling nanoparticles of Schiffelers et al.

The nanoplexes of Bartlett et al. (PNAS, Sep. 25, 2007, vol. 104, no.39) may also be applied to the present invention. The nanoplexes ofBartlett et al. are prepared by mixing equal volumes of aqueoussolutions of cationic polymer and nucleic acid to give a net molarexcess of ionizable nitrogen (polymer) to phosphate (nucleic acid) overthe range of 2 to 6. The electrostatic interactions between cationicpolymers and nucleic acid resulted in the formation of polyplexes withaverage particle size distribution of about 100 nm, hence referred tohere as nanoplexes. The DOTA-siRNA of Bartlett et al. was synthesized asfollows: 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acidmono(N-hydroxysuccinimide ester) (DOTA-NHSester) was ordered fromMacrocyclics (Dallas, Tex.). The amine modified RNA sense strand with a100-fold molar excess of DOTA-NHS-ester in carbonate buffer (pH 9) wasadded to a microcentrifuge tube. The contents were reacted by stirringfor 4 h at room temperature. The DOTA-RNAsense conjugate wasethanol-precipitated, resuspended in water, and annealed to theunmodified antisense strand to yield DOTA-siRNA. All liquids werepretreated with Chelex-100 (Bio-Rad, Hercules, Calif.) to remove tracemetal contaminants. Tf-targeted and nontargeted siRNA nanoparticles maybe formed by using cyclodextrin-containing polycations. Typically,nanoparticles were formed in water at a charge ratio of 3 (+/−) and ansiRNA concentration of 0.5 g/liter. One percent of the adamantane-PEGmolecules on the surface of the targeted nanoparticles were modifiedwith Tf (adamantane-PEG-Tf). The nanoparticles were suspended in a 5%(wt/vol) glucose carrier solution for injection.

Davis et al. (Nature, Vol 464, 15 Apr. 2010) conducts a RNA clinicaltrial that uses a targeted nanoparticle-delivery system (clinical trialregistration number NCT00689065). Patients with solid cancers refractoryto standard-of-care therapies are administered doses of targetednanoparticles on days 1, 3, 8 and 10 of a 21-day cycle by a 30-minintravenous infusion. The nanoparticles consist of a synthetic deliverysystem containing: (1) a linear, cyclodextrin-based polymer (CDP), (2) ahuman transferrin protein (TF) targeting ligand displayed on theexterior of the nanoparticle to engage TF receptors (TFR) on the surfaceof the cancer cells, (3) a hydrophilic polymer (polyethylene glycol(PEG) used to promote nanoparticle stability in biological fluids), and(4) siRNA designed to reduce the expression of the RRM2 (sequence usedin the clinic was previously denoted siR2B+5). The TFR has long beenknown to be upregulated in malignant cells, and RRM2 is an establishedanti-cancer target. These nanoparticles (clinical version denoted asCALAA-01) have been shown to be well tolerated in multi-dosing studiesin non-human primates. Although a single patient with chronic myeloidleukaemia has been administered siRNA by liposomal delivery, Davis etal.'s clinical trial is the initial human trial to systemically deliversiRNA with a targeted delivery system and to treat patients with solidcancer. To ascertain whether the targeted delivery system can provideeffective delivery of functional siRNA to human tumors, Davis et al.investigated biopsies from three patients from three different dosingcohorts; patients A, B and C, all of whom had metastatic melanoma andreceived CALAA-01 doses of 18, 24 and 30 mg m-2 siRNA, respectively.Similar doses may also be contemplated for the CRISPR Cas system of thepresent invention. The delivery of the invention may be achieved withnanoparticles containing a linear, cyclodextrin-based polymer (CDP), ahuman transferrin protein (TF) targeting ligand displayed on theexterior of the nanoparticle to engage TF receptors (TFR) on the surfaceof the cancer cells and/or a hydrophilic polymer (for example,polyethylene glycol (PEG) used to promote nanoparticle stability inbiological fluids).

U.S. Pat. No. 8,709,843, incorporated herein by reference, provides adrug delivery system for targeted delivery of therapeuticagent-containing particles to tissues, cells, and intracellularcompartments. The invention provides targeted particles comprisingcomprising polymer conjugated to a surfactant, hydrophilic polymer orlipid. U.S. Pat. No. 6,007,845, incorporated herein by reference,provides particles which have a core of a multiblock copolymer formed bycovalently linking a multifunctional compound with one or morehydrophobic polymers and one or more hydrophilic polymers, and conatin abiologically active material. U.S. Pat. No. 5,855,913, incorporatedherein by reference, provides a particulate composition havingaerodynamically light particles having a tap density of less than 0.4g/cm3 with a mean diameter of between 5 μm and 30 μm, incorporating asurfactant on the surface thereof for drug delivery to the pulmonarysystem. U.S. Pat. No. 5,985,309, incorporated herein by reference,provides particles incorporating a surfactant and/or a hydrophilic orhydrophobic complex of a positively or negatively charged therapeutic ordiagnostic agent and a charged molecule of opposite charge for deliveryto the pulmonary system. US. Pat. No. 5,543,158, incorporated herein byreference, provides biodegradable injectable particles having abiodegradable solid core containing a biologically active material andpoly(alkylene glycol) moieties on the surface. WO2012135025 (alsopublished as US20120251560), incorporated herein by reference, describesconjugated polyethyleneimine (PEI) polymers and conjugatedaza-macrocycles (collectively referred to as “conjugated lipomer” or“lipomers”). In certain embodiments, it can envisioned that suchconjugated lipomers can be used in the context of the CRISPR-Cas systemto achieve in vitro, ex vivo and in vivo genomic perturbations to modifygene expression, including modulation of protein expression.

In one embodiment, the nanoparticle may be epoxide-modifiedlipid-polymer, advantageously 7C1 (see, e.g., James E. Dahlman andCarmen Barnes et al. Nature Nanotechnology (2014) published online 11May 2014, doi:10.1038/nnano.2014.84). C71 was synthesized by reactingC15 epoxide-terminated lipids with PEI600 at a 14:1 molar ratio, and wasformulated with C14PEG2000 to produce nanoparticles (diameter between 35and 60 nm) that were stable in PBS solution for at least 40 days.

An epoxide-modified lipid-polymer may be utilized to deliver theCRISPR-Cas system of the present invention to pulmonary, cardiovascularor renal cells, however, one of skill in the art may adapt the system todeliver to other target organs. Dosage ranging from about 0.05 to about0.6 mg/kg are envisioned. Dosages over several days or weeks are alsoenvisioned, with a total dosage of about 2 mg/kg.

In some embodiments, the LNP for deliverting the RNA molecules isprepared by methods known in the art, such as those described in, forexample, WO 2005/105152 (PCT/EP2005/004920), WO 2006/069782(PCT/EP2005/014074), WO 2007/121947 (PCT/EP2007/003496), and WO2015/082080 (PCT/EP2014/003274), which are herein incorporated byreference. LNPs aimed specifically at the enhanced and improved deliveryof siRNA into mammalian cells are described in, for example, Aleku etal., Cancer Res., 68(23): 9788-98 (Dec. 1, 2008), Strumberg et al., Int.J. Clin. Pharmacol. Ther., 50(1): 76-8 (January 2012), Schultheis etal., J. Clin. Oncol., 32(36): 4141-48 (Dec. 20, 2014), and Fehring etal., Mol. Ther., 22(4): 811-20 (Apr. 22, 2014), which are hereinincorporated by reference and may be applied to the present technology.

In some embodiments, the LNP includes any LNP disclosed in WO2005/105152 (PCT/EP2005/004920), WO 2006/069782 (PCT/EP2005/014074), WO2007/121947 (PCT/EP2007/003496), and WO 2015/082080 (PCT/EP2014/003274).

In some embodiments, the LNP includes at least one lipid having FormulaI:

(Formula I), wherein R1 and R2 are each and independently selected fromthe group comprising alkyl, n is any integer between 1 and 4, and R3 isan acyl selected from the group comprising lysyl, ornithyl,2,4-diaminobutyryl, histidyl and an acyl moiety according to Formula II:

wherein m is any integer from 1 to 3 and Y⁻ is a pharmaceuticallyacceptable anion. In some embodiments, a lipid according to Formula Iincludes at least two asymmetric C atoms. In some embodiments,enantiomers of Formula I include, but are not limited to, R—R; S—S; R—Sand S—R enantiomer.

In some embodiments, R1 is lauryl and R2 is myristyl. In anotherembodiment, R1 is palmityl and R2 is oleyl. In some embodiments, m is 1or 2. In some embodiments, Y— is selected from halogenids, acetate ortrifluoroacetate.

In some embodiments, the LNP comprises one or more lipids select from:

arginyl-2,3-diamino propionic acid-N-palmityl-N-oleyl-amidetrihydrochloride (Formula III):

arginyl-2,3-diamino propionic acid-N-lauryl-N-myristyl-amidetrihydrochloride (Formula IV):

and

arginyl-lysine-N-lauryl-N-myristyl-amide trihydrochloride (Formula V):

In some embodiments, the LNP also includes a constituent. By way ofexample, but not by way of limitation, in some embodiments, theconstituent is selected from peptides, proteins, oligonucleotides,polynucleotides, nucleic acids, or a combination thereof. In someembodiments, the constituent is an antibody, e.g., a monoclonalantibody. In some embodiments, the constituent is a nucleic acidselected from, e.g., ribozymes, aptamers, spiegelmers, DNA, RNA, PNA,LNA, or a combination thereof. In some embodiments, the nucleic acid isguide RNA and/or mRNA.

In some embodiments, the constituent of the LNP comprises an mRNAencoding a CRIPSR-Cas protein. In some embodiments, the constituent ofthe LNP comprises an mRNA encoding a Type-II or Type-V CRIPSR-Casprotein. In some embodiments, the constituent of the LNP comprises anmRNA encoding an adenosine deaminase (which may be fused to a CRISPR-Casprotein or an adaptor protein).

In some embodiments, the constituent of the LNP further comprises one ormore guide RNA. In some embodiments, the LNP is configured to deliverthe aforementioned mRNA and guide RNA to vascular endothelium. In someembodiments, the LNP is configured to deliver the aforementioned mRNAand guide RNA to pulmonary endothelium. In some embodiments, the LNP isconfigured to deliver the aforementioned mRNA and guide RNA to liver. Insome embodiments, the LNP is configured to deliver the aforementionedmRNA and guide RNA to lung. In some embodiments, the LNP is configuredto deliver the aforementioned mRNA and guide RNA to hearts. In someembodiments, the LNP is configured to deliver the aforementioned mRNAand guide RNA to spleen. In some embodiments, the LNP is configured todeliver the aforementioned mRNA and guide RNA to kidney. In someembodiments, the LNP is configured to deliver the aforementioned mRNAand guide RNA to pancrea. In some embodiments, the LNP is configured todeliver the aforementioned mRNA and guide RNA to brain. In someembodiments, the LNP is configured to deliver the aforementioned mRNAand guide RNA to macrophages.

In some embodiments, the LNP also includes at least one helper lipid. Insome embodiments, the helper lipid is selected from phospholipids andsteroids. In some embodiments, the phospholipids are di- and/ormonoester of the phosphoric acid. In some embodiments, the phospholipidsare phosphoglycerides and/or sphingolipids. In some embodiments, thesteroids are naturally occurring and/or synthetic compounds based on thepartially hydrogenated cyclopenta[a]phenanthrene. In some embodiments,the steroids contain 21 to 30 C atoms. In some embodiments, the steroidis cholesterol. In some embodiments, the helper lipid is selected from1,2-diphytanoyl-sn-glycero-3-phosphoethanolamine (DPhyPE), ceramide, and1,2-dioleylsn-glycero-3-phosphoethanolamine (DOPE).

In some embodiments, the at least one helper lipid comprises a moietyselected from the group comprising a PEG moiety, a HEG moiety, apolyhydroxyethyl starch (polyHES) moiety and a polypropylene moiety. Insome embodiments, the moiety has a molecule weight between about 500 to10,000 Da or between about 2,000 to 5,000 Da. In some embodiments, thePEG moiety is selected from 1,2-distearoyl-sn-glycero-3phosphoethanolamine, 1,2-dialkyl-sn-glycero-3-phosphoethanolamine, andCeramide-PEG. In some embodiments, the PEG moiety has a molecular weightbetween about 500 to 10,000 Da or between about 2,000 to 5,000 Da. Insome embodiments, the PEG moiety has a molecular weight of 2,000 Da.

In some embodiments, the helper lipid is between about 20 mol % to 80mol % of the total lipid content of the composition. In someembodiments, the helper lipid component is between about 35 mol % to 65mol % of the total lipid content of the LNP. In some embodiments, theLNP includes lipids at 50 mol % and the helper lipid at 50 mol % of thetotal lipid content of the LNP.

In some embodiments, the LNP includes any of-3-arginyl-2,3-diaminopropionic acid-N-palmityl-N-oleyl-amidetrihydrochloride, -arginyl-2,3-diaminopropionicacid-N-lauryl-N-myristyl-amide trihydrochloride or-arginyl-lysine-N-lauryl-N-myristyl-amide trihydrochloride incombination with DPhyPE, wherein the content of DPhyPE is about 80 mol%, 65 mol %, 50 mol % and 35 mol % of the overall lipid content of theLNP. In some embodiments, the LNP includes -arginyl-2,3-diaminopropionic acid-N-pahnityl-N-oleyl-amide trihydrochloride (lipid) and1,2-diphytanoyl-sn-glycero-3-phosphoethanolamine (helper lipid). In someembodiments, the LNP includes -arginyl-2,3-diamino propionicacid-N-palmityl-N-oleyl-amide trihydrochloride (lipid),1,2-diphytanoyl-sn-glycero-3-phosphoethanolamine (first helper lipid),and 1,2-disteroyl-sn-glycero-3-phosphoethanolamine-PEG2000 (secondhelper lipid).

In some embodiments, the second helper lipid is between about 0.05 mol %to 4.9 mol % or between about 1 mol % to 3 mol % of the total lipidcontent. In some embodiments, the LNP includes lipids at between about45 mol % to 50 mol % of the total lipid content, a first helper lipidbetween about 45 mol % to 50 mol % of the total lipid content, under theproviso that there is a PEGylated second helper lipid between about 0.1mol % to 5 mol %, between about 1 mol % to 4 mol %, or at about 2 mol %of the total lipid content, wherein the sum of the content of thelipids, the first helper lipid, and of the second helper lipid is 100mol % of the total lipid content and wherein the sum of the first helperlipid and the second helper lipid is 50 mol % of the total lipidcontent. In some embodiments, the LNP comprises: (a) 50 mol % of-arginyl-2,3-diamino propionic acid-N-palmityl-N-oleyl-amidetrihydrochloride, 48 mol % of1,2-diphytanoyl-sn-glycero-3-phosphoethanolamine; and 2 mol %1,2-distearoyl-sn-glycero-3-phosphoethanolamine-PEG2000; or (b) 50 mol %of -arginyl-2,3-diamino propionic acid-N-palmityl-N-oleyl-amidetrihydrocloride, 49 mol %1,2-diphytanoyl-sn-glycero-3-phosphoethanolamine; and 1 mol %N(Carbonyl-methoxypolyethylenglycol-2000)-1,2-distearoyl-sn-glycero3-phosphoethanolamine,or a sodium salt thereof.

In some embodiments, the LNP contains a nucleic acid, wherein the chargeratio of nucleic acid backbone phosphates to cationic lipid nitrogenatoms is about 1: 1.5-7 or about 1:4.

In some embodiments, the LNP also includes a shielding compound, whichis removable from the lipid composition under in vivo conditions. Insome embodiments, the shielding compound is a biologically inertcompound. In some embodiments, the shielding compound does not carry anycharge on its surface or on the molecule as such. In some embodiments,the shielding compounds are polyethylenglycoles (PEGs),hydroxyethylglucose (HEG) based polymers, polyhydroxyethyl starch(polyHES) and polypropylene. In some embodiments, the PEG, HEG, polyHES,and a polypropylene weight between about 500 to 10,000 Da or betweenabout 2000 to 5000 Da. In some embodiments, the shielding compound isPEG2000 or PEG5000.

In some embodiments, the LNP includes at least one lipid, a first helperlipid, and a shielding compound that is removable from the lipidcomposition under in vivo conditions. In some embodiments, the LNP alsoincludes a second helper lipid. In some embodiments, the first helperlipid is ceramide. In some embodiments, the second helper lipid isceramide. In some embodiments, the ceramide comprises at least one shortcarbon chain substituent of from 6 to 10 carbon atoms. In someembodiments, the ceramide comprises 8 carbon atoms. In some embodiments,the shielding compound is attached to a ceramide. In some embodiments,the shielding compound is attached to a ceramide. In some embodiments,the shielding compound is covalently attached to the ceramide. In someembodiments, the shielding compound is attached to a nucleic acid in theLNP. In some embodiments, the shielding compound is covalently attachedto the nucleic acid. In some embodiments, the shielding compound isattached to the nucleic acid by a linker. In some embodiments, thelinker is cleaved under physiological conditions. In some embodiments,the linker is selected from ssRNA, ssDNA, dsRNA, dsDNA, peptide,S-S-linkers and pH sensitive linkers. In some embodiments, the linkermoiety is attached to the 3′ end of the sense strand of the nucleicacid. In some embodiments, the shielding compound comprises apH-sensitive linker or a pH-sensitive moiety. In some embodiments, thepH-sensitive linker or pH-sensitive moiety is an anionic linker or ananionic moiety. In some embodiments, the anionic linker or anionicmoiety is less anionic or neutral in an acidic environment. In someembodiments, the pH-sensitive linker or the pH-sensitive moiety isselected from the oligo (glutamic acid), oligophenolate(s) anddiethylene triamine penta acetic acid.

In any of the LNP embodiments in the previous paragraph, the LNP canhave an osmolality between about 50 to 600 mosmole/kg, between about 250to 350 mosmole/kg, or between about 280 to 320 mosmole/kg, and/orwherein the LNP formed by the lipid and/or one or two helper lipids andthe shielding compound have a particle size between about 20 to 200 nm,between about 30 to 100 nm, or between about 40 to 80 nm.

In some embodiments, the shielding compound provides for a longercirculation time in vivo and allows for a better biodistribution of thenucleic acid containing LNP. In some embodiments, the shielding compoundprevents immediate interaction of the LNP with serum compounds orcompounds of other bodily fluids or cytoplasma membranes, e.g.,cytoplasma membranes of the endothelial lining of the vasculature, intowhich the LNP is administered. Additionally or alternatively, in someembodiments, the shielding compounds also prevent elements of the immunesystem from immediately interacting with the LNP. Additionally oralternatively, in some embodiments, the shielding compound acts as ananti-opsonizing compound. Without wishing to be bound by any mechanismor theory, in some embodiments, the shielding compound forms a cover orcoat that reduces the surface area of the LNP available for interactionwith its environment. Additionally or alternatively, in someembodiments, the shielding compound shields the overall charge of theLNP.

In another embodiment, the LNP includes at least one cationic lipidhaving Formula VI:

wherein n is 1, 2, 3, or 4, wherein m is 1, 2, or 3, wherein Y⁻ isanion, wherein each of 10 and R² is individually and independentlyselected from the group consisting of linear C12-C18 alkyl and linearC12-C18 alkenyl, a sterol compound, wherein the sterol compound isselected from the group consisting of cholesterol and stigmasterol, anda PEGylated lipid, wherein the PEGylated lipid comprises a PEG moiety,wherein the PEGylated lipid is selected from the group consisting of:

-   a PEGylated phosphoethanolamine of Formula VII:

wherein R³ and R⁴ are individually and independently linear C13-C17alkyl, and p is any integer between 15 to 130;

-   a PEGylated ceramide of Formula VIII:

wherein R⁵ is linear C7-C15 alkyl, and q is any number between 15 to130; and

-   a PEGylated diacylglycerol of Formula IX:

wherein each of R⁶ and R⁷ is individually and independently linearC11-C17 alkyl, and r is any integer from 15 to 130.

In some embodiments, R¹ and R² are different from each other. In someembodiments, R¹ is palmityl and R² is oleyl. In some embodiments, R¹ islauryl and R² is myristyl. In some embodiments, R¹ and R² are the same.In some embodiments, each of R¹ and R² is individually and independentlyselected from the group consisting of C12 alkyl, C14 alkyl, C16 alkyl,C18 alkyl, C12 alkenyl, C14 alkenyl, C16 alkenyl and C18 alkenyl. Insome embodiments, each of C12 alkenyl, C14 alkenyl, C16 alkenyl and Cl 8alkenyl comprises one or two double bonds. In some embodiments, C18alkenyl is C18 alkenyl with one double bond between C9 and C10. In someembodiments, C18 alkenyl is cis-9-octadecyl.

In some embodiments, the cationic lipid is a compound of Formula X:

In some embodiments, Y⁻ is selected from halogenids, acetate andtrifluoroacetate. In some embodiments, the cationic lipid is-arginyl-2,3-diamino propionic acid-N-palmityl-N-oleyl-amidetrihydrochloride of Formula III:

In some embodiments, the cationic lipid is -arginyl-2,3-diaminopropionic acid-N-lauryl-N-myristyl-amide trihydrochloride of Formula IV:

In some embodiments, the cationic lipid is-arginyl-lysine-N-lauryl-N-myristyl-amide trihydrochloride of Formula V:

In some embodiments, the sterol compound is cholesterol. In someembodiments, the sterol compound is stigmasterin.

In some embodiments, the PEG moiety of the PEGylated lipid has amolecular weight from about 800 to 5,000 Da. In some embodiments, themolecular weight of the PEG moiety of the PEGylated lipid is about 800Da. In some embodiments, the molecular weight of the PEG moiety of thePEGylated lipid is about 2,000 Da. In some embodiments, the molecularweight of the PEG moiety of the PEGylated lipid is about 5,000 Da. Insome embodiments, the PEGylated lipid is a PEGylated phosphoethanolamineof Formula VII, wherein each of R³ and R⁴ is individually andindependently linear C13-C17 alkyl, and p is any integer from 18, 19 or20, or from 44, 45 or 46 or from 113, 114 or 115. In some embodiments,R³ and R⁴ are the same. In some embodiments, R³ and R⁴ are different. Insome embodiments, each of R³ and R⁴ is individually and independentlyselected from the group consisting of C13 alkyl, C15 alkyl and C17alkyl. In some embodiments, the PEGylated phosphoethanolamine of FormulaVII is1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethyleneglycol)-2000] (ammonium salt):

In some embodiments, the PEGylated phosphoethanolamine of Formula VII is1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethyleneglycol)-5000] (ammonium salt):

In some embodiments, the PEGylated lipid is a PEGylated ceramide ofFormula VIII, wherein R⁵ is linear C7-C15 alkyl, and q is any integerfrom 18, 19 or 20, or from 44, 45 or 46 or from 113, 114 or 115. In someembodiments, R⁵ is linear C7 alkyl. In some embodiments, R⁵ is linearC15 alkyl. In some embodiments, the PEGylated ceramide of Formula VIIIis N-octanoyl-sphingosine-1-{succinyl [methoxy(polyethyleneglycol)2000]}:

In some embodiments, the PEGylated ceramide of Formula VIII isN-palmitoyl-sphingosine-1-{succinyl[methoxy(polyethylene glycol)2000]}

In some embodiments, the PEGylated lipid is a PEGylated diacylglycerolof Formula IX, wherein each of R⁶ and R⁷ is individually andindependently linear C11-C17 alkyl, and r is any integer from 18, 19 or20, or from 44, 45 or 46 or from 113, 114 or 115. In some embodiments,R⁶ and R⁷ are the same. In some embodiments, R⁶ and R⁷ are different. Insome embodiments, each of R⁶ and R⁷ is individually and independentlyselected from the group consisting of linear C17 alkyl, linear C15 alkyland linear C13 alkyl. In some embodiments, the PEGylated diacylglycerolof Formula IX 1,2-Distearoyl-sn-glycerol [methoxy(polyethyleneglycol)2000]:

In some embodiments, the PEGylated diacylglycerol of Formula IX is1,2-Dipalmitoyl-sn-glycerol [methoxy(polyethylene glycol)2000]:

In some embodiments, the PEGylated diacylglycerol of Formula IX is:

In some embodiments, the LNP includes at least one cationic lipidselected from of Formulas III, IV, and V, at least one sterol compoundselected from a cholesterol and stigmasterin, and wherein the PEGylatedlipid is at least one selected from Formulas XI and XII. In someembodiments, the LNP includes at least one cationic lipid selected fromFormulas III, IV, and V, at least one sterol compound selected from acholesterol and stigmasterin, and wherein the PEGylated lipid is atleast one selected from Formulas XIII and XIV. In some embodiments, theLNP includes at least one cationic lipid selected from Formulas III, IV,and V, at least one sterol compound selected from a cholesterol andstigmasterin, and wherein the PEGylated lipid is at least one selectedfrom Formulas XV and XVI. In some embodiments, the LNP includes acationic lipid of Formula III, a cholesterol as the sterol compound, andwherein the PEGylated lipid is Formula XI.

In any of the LNP embodiments in the previous paragraph, wherein thecontent of the cationic lipid composition is between about 65 mole % to75 mole %, the content of the sterol compound is between about 24 mole %to 34 mole % and the content of the PEGylated lipid is between about 0.5mole % to 1.5 mole %, wherein the sum of the content of the cationiclipid, of the sterol compound and of the PEGylated lipid for the lipidcomposition is 100 mole %. In some embodiments, the cationic lipid isabout 70 mole %, the content of the sterol compound is about 29 mole %and the content of the PEGylated lipid is about 1 mole %. In someembodiments, the LNP is 70 mole % of Formula III, 29 mole % ofcholesterol, and 1 mole % of Formula XI.

Exosomes

Exosomes are endogenous nano-vesicles that transport RNAs and proteins,and which can deliver RNA to the brain and other target organs. Toreduce immunogenicity, Alvarez-Erviti et al. (2011, Nat Biotechnol 29:341) used self-derived dendritic cells for exosome production. Targetingto the brain was achieved by engineering the dendritic cells to expressLamp2b, an exosomal membrane protein, fused to the neuron-specific RVGpeptide. Purified exosomes were loaded with exogenous RNA byelectroporation. Intravenously injected RVG-targeted exosomes deliveredGAPDH siRNA specifically to neurons, microglia, oligodendrocytes in thebrain, resulting in a specific gene knockdown. Pre-exposure to RVGexosomes did not attenuate knockdown, and non-specific uptake in othertissues was not observed. The therapeutic potential of exosome-mediatedsiRNA delivery was demonstrated by the strong mRNA (60%) and protein(62%) knockdown of BACE1, a therapeutic target in Alzheimer's disease.

To obtain a pool of immunologically inert exosomes, Alvarez-Erviti etal. harvested bone marrow from inbred C57BL/6 mice with a homogenousmajor histocompatibility complex (MHC) haplotype. As immature dendriticcells produce large quantities of exosomes devoid of T-cell activatorssuch as MHC-II and CD86, Alvarez-Erviti et al. selected for dendriticcells with granulocyte/macrophage-colony stimulating factor (GM-CSF) for7 d. Exosomes were purified from the culture supernatant the followingday using well-established ultracentrifugation protocols. The exosomesproduced were physically homogenous, with a size distribution peaking at80 nm in diameter as determined by nanoparticle tracking analysis (NTA)and electron microscopy. Alvarez-Erviti et al. obtained 6-12 μg ofexosomes (measured based on protein concentration) per 106 cells.

Next, Alvarez-Erviti et al. investigated the possibility of loadingmodified exosomes with exogenous cargoes using electroporation protocolsadapted for nanoscale applications. As electroporation for membraneparticles at the nanometer scale is not well-characterized, nonspecificCy5-labeled RNA was used for the empirical optimization of theelectroporation protocol. The amount of encapsulated RNA was assayedafter ultracentrifugation and lysis of exosomes. Electroporation at 400V and 125 μF resulted in the greatest retention of RNA and was used forall subsequent experiments.

Alvarez-Erviti et al. administered 150 μg of each BACE1 siRNAencapsulated in 150 μg of RVG exosomes to normal C57BL/6 mice andcompared the knockdown efficiency to four controls: untreated mice, miceinjected with RVG exosomes only, mice injected with BACE1 siRNAcomplexed to an in vivo cationic liposome reagent and mice injected withBACE1 siRNA complexed to RVG-9R, the RVG peptide conjugated to 9D-arginines that electrostatically binds to the siRNA. Cortical tissuesamples were analyzed 3 d after administration and a significant proteinknockdown (45%, P<0.05, versus 62%, P<0.01) in both siRNA-RVG-9R-treatedand siRNARVG exosome-treated mice was observed, resulting from asignificant decrease in BACE1 mRNA levels (66% [+ or −] 15%, P<0.001 and61% [+ or −] 13% respectively, P<0.01). Moreover, Applicantsdemonstrated a significant decrease (55%, P<0.05) in the total[beta]-amyloid 1-42 levels, a main component of the amyloid plaques inAlzheimer's pathology, in the RVG-exosome-treated animals. The decreaseobserved was greater than the β-amyloid 1-40 decrease demonstrated innormal mice after intraventricular injection of BACE1 inhibitors.Alvarez-Erviti et al. carried out 5′-rapid amplification of cDNA ends(RACE) on BACE1 cleavage product, which provided evidence ofRNAi-mediated knockdown by the siRNA.

Finally, Alvarez-Erviti et al. investigated whether RNA-RVG exosomesinduced immune responses in vivo by assessing IL-6, IP-10, TNFα andIFN-α serum concentrations. Following exosome treatment, nonsignificantchanges in all cytokines were registered similar to siRNA-transfectionreagent treatment in contrast to siRNA-RVG-9R, which potently stimulatedIL-6 secretion, confirming the immunologically inert profile of theexosome treatment. Given that exosomes encapsulate only 20% of siRNA,delivery with RVG-exosome appears to be more efficient than RVG-9Rdelivery as comparable mRNA knockdown and greater protein knockdown wasachieved with fivefold less siRNA without the corresponding level ofimmune stimulation. This experiment demonstrated the therapeuticpotential of RVG-exosome technology, which is potentially suited forlong-term silencing of genes related to neurodegenerative diseases. Theexosome delivery system of Alvarez-Erviti et al. may be applied todeliver the AD-functionalized CRISPR-Cas system of the present inventionto therapeutic targets, especially neurodegenerative diseases. A dosageof about 100 to 1000 mg of CRISPR Cas encapsulated in about 100 to 1000mg of RVG exosomes may be contemplated for the present invention.

El-Andaloussi et al. (Nature Protocols 7, 2112-2126(2012)) discloses howexosomes derived from cultured cells can be harnessed for delivery ofRNA in vitro and in vivo. This protocol first describes the generationof targeted exosomes through transfection of an expression vector,comprising an exosomal protein fused with a peptide ligand. Next,El-Andaloussi et al. explain how to purify and characterize exosomesfrom transfected cell supernatant. Next, El-Andaloussi et al. detailcrucial steps for loading RNA into exosomes. Finally, El-Andaloussi etal. outline how to use exosomes to efficiently deliver RNA in vitro andin vivo in mouse brain. Examples of anticipated results in whichexosome-mediated RNA delivery is evaluated by functional assays andimaging are also provided. The entire protocol takes ˜3 weeks. Deliveryor administration according to the invention may be performed usingexosomes produced from self-derived dendritic cells. From the hereinteachings, this can be employed in the practice of the invention.

In another embodiment, the plasma exosomes of Wahlgren et al. (NucleicAcids Research, 2012, Vol. 40, No. 17 e130) are contemplated. Exosomesare nano-sized vesicles (30-90 nm in size) produced by many cell types,including dendritic cells (DC), B cells, T cells, mast cells, epithelialcells and tumor cells. These vesicles are formed by inward budding oflate endosomes and are then released to the extracellular environmentupon fusion with the plasma membrane. Because exosomes naturally carryRNA between cells, this property may be useful in gene therapy, and fromthis disclosure can be employed in the practice of the instantinvention.

Exosomes from plasma can be prepared by centrifugation of buffy coat at900 g for 20 min to isolate the plasma followed by harvesting cellsupernatants, centrifuging at 300 g for 10 min to eliminate cells and at16 500 g for 30 min followed by filtration through a 0.22 mm filter.Exosomes are pelleted by ultracentrifugation at 120 000 g for 70 min.Chemical transfection of siRNA into exosomes is carried out according tothe manufacturer's instructions in RNAi Human/Mouse Starter Kit(Quiagen, Hilden, Germany). siRNA is added to 100 ml PBS at a finalconcentration of 2 mmol/ml. After adding HiPerFect transfection reagent,the mixture is incubated for 10 min at RT. In order to remove the excessof micelles, the exosomes are re-isolated using aldehyde/sulfate latexbeads. The chemical transfection of CRISPR Cas into exosomes may beconducted similarly to siRNA. The exosomes may be co-cultured withmonocytes and lymphocytes isolated from the peripheral blood of healthydonors. Therefore, it may be contemplated that exosomes containingCRISPR Cas may be introduced to monocytes and lymphocytes of andautologously reintroduced into a human. Accordingly, delivery oradministration according to the invention may be performed using plasmaexosomes.

Liposomes

Delivery or administration according to the invention can be performedwith liposomes. Liposomes are spherical vesicle structures composed of auni- or multilamellar lipid bilayer surrounding internal aqueouscompartments and a relatively impermeable outer lipophilic phospholipidbilayer. Liposomes have gained considerable attention as drug deliverycarriers because they are biocompatible, nontoxic, can deliver bothhydrophilic and lipophilic drug molecules, protect their cargo fromdegradation by plasma enzymes, and transport their load acrossbiological membranes and the blood brain barrier (BBB) (see, e.g., Spuchand Navarro, Journal of Drug Delivery, vol. 2011, Article ID 469679, 12pages, 2011. doi:10.1155/2011/469679 for review).

Liposomes can be made from several different types of lipids; however,phospholipids are most commonly used to generate liposomes as drugcarriers. Although liposome formation is spontaneous when a lipid filmis mixed with an aqueous solution, it can also be expedited by applyingforce in the form of shaking by using a homogenizer, sonicator, or anextrusion apparatus (see, e.g., Spuch and Navarro, Journal of DrugDelivery, vol. 2011, Article ID 469679, 12 pages, 2011.doi:10.1155/2011/469679 for review).

Several other additives may be added to liposomes in order to modifytheir structure and properties. For instance, either cholesterol orsphingomyelin may be added to the liposomal mixture in order to helpstabilize the liposomal structure and to prevent the leakage of theliposomal inner cargo. Further, liposomes are prepared from hydrogenatedegg phosphatidylcholine or egg phosphatidylcholine, cholesterol, anddicetyl phosphate, and their mean vesicle sizes were adjusted to about50 and 100 nm. (see, e.g., Spuch and Navarro, Journal of Drug Delivery,vol. 2011, Article ID 469679, 12 pages, 2011. doi:10.1155/2011/469679for review).

A liposome formulation may be mainly comprised of natural phospholipidsand lipids such as 1,2-distearoryl-sn-glycero-3-phosphatidyl choline(DSPC), sphingomyelin, egg phosphatidylcholines andmonosialoganglioside. Since this formulation is made up of phospholipidsonly, liposomal formulations have encountered many challenges, one ofthe ones being the instability in plasma. Several attempts to overcomethese challenges have been made, specifically in the manipulation of thelipid membrane. One of these attempts focused on the manipulation ofcholesterol. Addition of cholesterol to conventional formulationsreduces rapid release of the encapsulated bioactive compound into theplasma or 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE) increasesthe stability (see, e.g., Spuch and Navarro, Journal of Drug Delivery,vol. 2011, Article ID 469679, 12 pages, 2011. doi:10.1155/2011/469679for review).

In a particularly advantageous embodiment, Trojan Horse liposomes (alsoknown as Molecular Trojan Horses) are desirable and protocols may befound at cshprotocols.cshlp.org/content/2010/4/pdb.prot5407.long. Theseparticles allow delivery of a transgene to the entire brain after anintravascular injection. Without being bound by limitation, it isbelieved that neutral lipid particles with specific antibodiesconjugated to surface allow crossing of the blood brain barrier viaendocytosis. Trojan Horse Liposomes may be used to deliver the CRISPRfamily of nucleases to the brain via an intravascular injection, whichwould allow whole brain transgenic animals without the need forembryonic manipulation. About 1-5 g of DNA or RNA may be contemplatedfor in vivo administration in liposomes.

In another embodiment, the AD-functionalized CRISPR Cas system orcomponents thereof may be administered in liposomes, such as a stablenucleic-acid-lipid particle (SNALP) (see, e.g., Morrissey et al., NatureBiotechnology, Vol. 23, No. 8, August 2005). Daily intravenousinjections of about 1, 3 or 5 mg/kg/day of a specific CRISPR Castargeted in a SNALP are contemplated. The daily treatment may be overabout three days and then weekly for about five weeks. In anotherembodiment, a specific CRISPR Cas encapsulated SNALP) administered byintravenous injection to at doses of about 1 or 2.5 mg/kg are alsocontemplated (see, e.g., Zimmerman et al., Nature Letters, Vol. 441, 4May 2006). The SNALP formulation may contain the lipids3-N-[(wmethoxypoly(ethylene glycol) 2000)carbamoyl]-1,2-dimyristyloxy-propylamine (PEG-C-DMA),1,2-dilinoleyloxy-N,N-dimethyl-3-aminopropane (DLinDMA),1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC) and cholesterol, in a2:40:10:48 molar percent ratio (see, e.g., Zimmerman et al., NatureLetters, Vol. 441, 4 May 2006).

In another embodiment, stable nucleic-acid-lipid particles (SNALPs) haveproven to be effective delivery molecules to highly vascularizedHepG2-derived liver tumors but not in poorly vascularized HCT-116derived liver tumors (see, e.g., Li, Gene Therapy (2012) 19, 775-780).The SNALP liposomes may be prepared by formulating D-Lin-DMA andPEG-C-DMA with distearoylphosphatidylcholine (DSPC), Cholesterol andsiRNA using a 25:1 lipid/siRNA ratio and a 48/40/10/2 molar ratio ofCholesterol/D-Lin-DMA/DSPC/PEG-C-DMA. The resulted SNALP liposomes areabout 80-100 nm in size.

In yet another embodiment, a SNALP may comprise synthetic cholesterol(Sigma-Aldrich, St Louis, Mo., USA), dipalmitoylphosphatidylcholine(Avanti Polar Lipids, Alabaster, Ala., USA), 3-N-[(w-methoxypoly(ethylene glycol)2000)carbamoyl]-1,2-dimyrestyloxypropylamine, andcationic 1,2-dilinoleyloxy-3-N,Ndimethylaminopropane (see, e.g.,Geisbert et al., Lancet 2010; 375: 1896-905). A dosage of about 2 mg/kgtotal CRISPR Cas per dose administered as, for example, a bolusintravenous infusion may be contemplated.

In yet another embodiment, a SNALP may comprise synthetic cholesterol(Sigma-Aldrich), 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC;Avanti Polar Lipids Inc.), PEG-cDMA, and1,2-dilinoleyloxy-3-(N;N-dimethyl)aminopropane (DLinDMA) (see, e.g.,Judge, J. Clin. Invest. 119:661-673 (2009)). Formulations used for invivo studies may comprise a final lipid/RNA mass ratio of about 9:1.

The safety profile of RNAi nanomedicines has been reviewed by Barros andGollob of Alnylam Pharmaceuticals (see, e.g., Advanced Drug DeliveryReviews 64 (2012) 1730-1737). The stable nucleic acid lipid particle(SNALP) is comprised of four different lipids—an ionizable lipid(DLinDMA) that is cationic at low pH, a neutral helper lipid,cholesterol, and a diffusible polyethylene glycol (PEG)-lipid. Theparticle is approximately 80 nm in diameter and is charge-neutral atphysiologic pH. During formulation, the ionizable lipid serves tocondense lipid with the anionic RNA during particle formation. Whenpositively charged under increasingly acidic endosomal conditions, theionizable lipid also mediates the fusion of SNALP with the endosomalmembrane enabling release of RNA into the cytoplasm. The PEG-lipidstabilizes the particle and reduces aggregation during formulation, andsubsequently provides a neutral hydrophilic exterior that improvespharmacokinetic properties.

To date, two clinical programs have been initiated using SNALPformulations with RNA. Tekmira Pharmaceuticals recently completed aphase I single-dose study of SNALP-ApoB in adult volunteers withelevated LDL cholesterol. ApoB is predominantly expressed in the liverand jejunum and is essential for the assembly and secretion of VLDL andLDL. Seventeen subjects received a single dose of SNALP-ApoB (doseescalation across 7 dose levels). There was no evidence of livertoxicity (anticipated as the potential dose-limiting toxicity based onpreclinical studies). One (of two) subjects at the highest doseexperienced flu-like symptoms consistent with immune system stimulation,and the decision was made to conclude the trial.

Alnylam Pharmaceuticals has similarly advanced ALN-TTR01, which employsthe SNALP technology described above and targets hepatocyte productionof both mutant and wild-type TTR to treat TTR amyloidosis (ATTR). ThreeATTR syndromes have been described: familial amyloidotic polyneuropathy(FAP) and familial amyloidotic cardiomyopathy (FAC)—both caused byautosomal dominant mutations in TTR; and senile systemic amyloidosis(SSA) cause by wildtype TTR. A placebo-controlled, singledose-escalation phase I trial of ALN-TTR01 was recently completed inpatients with ATTR. ALN-TTR01 was administered as a 15-minute IVinfusion to 31 patients (23 with study drug and 8 with placebo) within adose range of 0.01 to 1.0 mg/kg (based on siRNA). Treatment was welltolerated with no significant increases in liver function tests.Infusion-related reactions were noted in 3 of 23 patients at ≥0.4 mg/kg;all responded to slowing of the infusion rate and all continued onstudy. Minimal and transient elevations of serum cytokines IL-6, IP-10and IL-1ra were noted in two patients at the highest dose of 1 mg/kg (asanticipated from preclinical and NHP studies). Lowering of serum TTR,the expected pharmacodynamics effect of ALN-TTR01, was observed at 1mg/kg.

In yet another embodiment, a SNALP may be made by solubilizing acationic lipid, DSPC, cholesterol and PEG-lipid e.g., in ethanol, e.g.,at a molar ratio of 40:10:40:10, respectively (see, Semple et al.,Nature Niotechnology, Volume 28 Number 2 February 2010, pp. 172-177).The lipid mixture was added to an aqueous buffer (50 mM citrate, pH 4)with mixing to a final ethanol and lipid concentration of 30% (vol/vol)and 6.1 mg/ml, respectively, and allowed to equilibrate at 22° C. for 2min before extrusion. The hydrated lipids were extruded through twostacked 80 nm pore-sized filters (Nuclepore) at 22° C. using a LipexExtruder (Northern Lipids) until a vesicle diameter of 70-90 nm, asdetermined by dynamic light scattering analysis, was obtained. Thisgenerally required 1-3 passes. The siRNA (solubilized in a 50 mMcitrate, pH 4 aqueous solution containing 30% ethanol) was added to thepre-equilibrated (35° C.) vesicles at a rate of ˜5 ml/min with mixing.After a final target siRNA/lipid ratio of 0.06 (wt/wt) was reached, themixture was incubated for a further 30 min at 35° C. to allow vesiclereorganization and encapsulation of the siRNA. The ethanol was thenremoved and the external buffer replaced with PBS (155 mM NaCl, 3 mMNa2HPO4, 1 mM KH2PO4, pH 7.5) by either dialysis or tangential flowdiafiltration. siRNA were encapsulated in SNALP using a controlledstep-wise dilution method process. The lipid constituents of KC2-SNALPwere DLin-KC2-DMA (cationic lipid), dipalmitoylphosphatidylcholine(DPPC; Avanti Polar Lipids), synthetic cholesterol (Sigma) and PEG-C-DMAused at a molar ratio of 57.1:7.1:34.3:1.4. Upon formation of the loadedparticles, SNALP were dialyzed against PBS and filter sterilized througha 0.2 μm filter before use. Mean particle sizes were 75-85 nm and 90-95%of the siRNA was encapsulated within the lipid particles. The finalsiRNA/lipid ratio in formulations used for in vivo testing was ˜0.15(wt/wt). LNP-siRNA systems containing Factor VII siRNA were diluted tothe appropriate concentrations in sterile PBS immediately before use andthe formulations were administered intravenously through the lateraltail vein in a total volume of 10 ml/kg. This method and these deliverysystems may be extrapolated to the AD-functionalized CRISPR Cas systemof the present invention.

Other Lipids

Other cationic lipids, such as amino lipid2,2-dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane (DLin-KC2-DMA) maybe utilized to encapsulate CRISPR Cas or components thereof or nucleicacid molecule(s) coding therefor e.g., similar to SiRNA (see, e.g.,Jayaraman, Angew. Chem. Int. Ed. 2012, 51, 8529-8533), and hence may beemployed in the practice of the invention. A preformed vesicle with thefollowing lipid composition may be contemplated: amino lipid, distearoylphosphatidylcholine (DSPC), cholesterol and(R)-2,3-bis(octadecyloxy) propyl-1-(methoxy poly(ethyleneglycol)2000)propylcarbamate (PEG-lipid) in the molar ratio 40/10/40/10,respectively, and a FVII siRNA/total lipid ratio of approximately 0.05(w/w). To ensure a narrow particle size distribution in the range of70-90 nm and a low polydispersity index of 0.11+0.04 (n=56), theparticles may be extruded up to three times through 80 nm membranesprior to adding the guide RNA. Particles containing the highly potentamino lipid 16 may be used, in which the molar ratio of the four lipidcomponents 16, DSPC, cholesterol and PEG-lipid (50/10/38.5/1.5) whichmay be further optimized to enhance in vivo activity.

Michael S D Kormann et al. (“Expression of therapeutic proteins afterdelivery of chemically modified mRNA in mice: Nature Biotechnology,Volume: 29, Pages: 154-157 (2011)) describes the use of lipid envelopesto deliver RNA. Use of lipid envelopes is also preferred in the presentinvention.

In another embodiment, lipids may be formulated with theAD-functionalized CRISPR Cas system of the present invention orcomponent(s) thereof or nucleic acid molecule(s) coding therefor to formlipid nanoparticles (LNPs). Lipids include, but are not limited to,DLin-KC2-DMA4, C12-200 and colipids disteroylphosphatidyl choline,cholesterol, and PEG-DMG may be formulated with CRISPR Cas instead ofsiRNA (see, e.g., Novobrantseva, Molecular Therapy-Nucleic Acids (2012)1, e4; doi:10.1038/mtna.2011.3) using a spontaneous vesicle formationprocedure. The component molar ratio may be about 50/10/38.5/1.5(DLin-KC2-DMA or C12-200/di steroylphosphatidylcholine/cholesterol/PEG-DMG). The final lipid: siRNA weight ratio may be˜12:1 and 9:1 in the case of DLin-KC2-DMA and C12-200 lipidnanoparticles (LNPs), respectively. The formulations may have meanparticle diameters of ˜80 nm with >90% entrapment efficiency. A 3 mg/kgdose may be contemplated.

Tekmira has a portfolio of approximately 95 patent families, in the U.S.and abroad, that are directed to various aspects of LNPs and LNPformulations (see, e.g., U.S. Pat. Nos. 7,982,027; 7,799,565; 8,058,069;8,283,333; 7,901,708; 7,745,651; 7,803,397; 8,101,741; 8,188,263;7,915,399; 8,236,943 and 7,838,658 and European Pat. Nos 1766035;1519714; 1781593 and 1664316), all of which may be used and/or adaptedto the present invention.

The AD-functionalized CRISPR Cas system or components thereof or nucleicacid molecule(s) coding therefor may be delivered encapsulated in PLGAMicrospheres such as that further described in US published applications20130252281 and 20130245107 and 20130244279 (assigned to ModernaTherapeutics) which relate to aspects of formulation of compositionscomprising modified nucleic acid molecules which may encode a protein, aprotein precursor, or a partially or fully processed form of the proteinor a protein precursor. The formulation may have a molar ratio50:10:38.5:1.5-3.0 (cationic lipid:fusogenic lipid:cholesterol:PEGlipid). The PEG lipid may be selected from, but is not limited toPEG-c-DOMG, PEG-DMG. The fusogenic lipid may be DSPC. See also, Schrumet al., Delivery and Formulation of Engineered Nucleic Acids, USpublished application 20120251618.

Nanomerics' technology addresses bioavailability challenges for a broadrange of therapeutics, including low molecular weight hydrophobic drugs,peptides, and nucleic acid based therapeutics (plasmid, siRNA, miRNA).Specific administration routes for which the technology has demonstratedclear advantages include the oral route, transport across theblood-brain-barrier, delivery to solid tumours, as well as to the eye.See, e.g., Mazza et al., 2013, ACS Nano. 2013 Feb. 26; 7(2):1016-26;Uchegbu and Siew, 2013, J Pharm Sci. 102(2):305-10 and Lalatsa et al.,2012, J Control Release. 2012 Jul. 20; 161(2):523-36.

US Patent Publication No. 20050019923 describes cationic dendrimers fordelivering bioactive molecules, such as polynucleotide molecules,peptides and polypeptides and/or pharmaceutical agents, to a mammalianbody. The dendrimers are suitable for targeting the delivery of thebioactive molecules to, for example, the liver, spleen, lung, kidney orheart (or even the brain). Dendrimers are synthetic 3-dimensionalmacromolecules that are prepared in a step-wise fashion from simplebranched monomer units, the nature and functionality of which can beeasily controlled and varied. Dendrimers are synthesised from therepeated addition of building blocks to a multifunctional core(divergent approach to synthesis), or towards a multifunctional core(convergent approach to synthesis) and each addition of a 3-dimensionalshell of building blocks leads to the formation of a higher generationof the dendrimers. Polypropylenimine dendrimers start from adiaminobutane core to which is added twice the number of amino groups bya double Michael addition of acrylonitrile to the primary aminesfollowed by the hydrogenation of the nitriles. This results in adoubling of the amino groups. Polypropylenimine dendrimers contain 100%protonable nitrogens and up to 64 terminal amino groups (generation 5,DAB 64). Protonable groups are usually amine groups which are able toaccept protons at neutral pH. The use of dendrimers as gene deliveryagents has largely focused on the use of the polyamidoamine. andphosphorous containing compounds with a mixture of amine/amide orN—P(O2)S as the conjugating units respectively with no work beingreported on the use of the lower generation polypropylenimine dendrimersfor gene delivery. Polypropylenimine dendrimers have also been studiedas pH sensitive controlled release systems for drug delivery and fortheir encapsulation of guest molecules when chemically modified byperipheral amino acid groups. The cytotoxicity and interaction ofpolypropylenimine dendrimers with DNA as well as the transfectionefficacy of DAB 64 has also been studied.

US Patent Publication No. 20050019923 is based upon the observationthat, contrary to earlier reports, cationic dendrimers, such aspolypropylenimine dendrimers, display suitable properties, such asspecific targeting and low toxicity, for use in the targeted delivery ofbioactive molecules, such as genetic material. In addition, derivativesof the cationic dendrimer also display suitable properties for thetargeted delivery of bioactive molecules. See also, Bioactive Polymers,US published application 20080267903, which discloses “Various polymers,including cationic polyamine polymers and dendrimeric polymers, areshown to possess anti-proliferative activity, and may therefore beuseful for treatment of disorders characterised by undesirable cellularproliferation such as neoplasms and tumours, inflammatory disorders(including autoimmune disorders), psoriasis and atherosclerosis. Thepolymers may be used alone as active agents, or as delivery vehicles forother therapeutic agents, such as drug molecules or nucleic acids forgene therapy. In such cases, the polymers' own intrinsic anti-tumoractivity may complement the activity of the agent to be delivered.” Thedisclosures of these patent publications may be employed in conjunctionwith herein teachings for delivery of AD-functionalized CRISPR Cassystem(s) or component(s) thereof or nucleic acid molecule(s) codingtherefor.

Supercharged Proteins

Supercharged proteins are a class of engineered or naturally occurringproteins with unusually high positive or negative net theoretical chargeand may be employed in delivery of AD-functionalized CRISPR Cassystem(s) or component(s) thereof or nucleic acid molecule(s) codingtherefor. Both supernegatively and superpositively charged proteinsexhibit a remarkable ability to withstand thermally or chemicallyinduced aggregation. Superpositively charged proteins are also able topenetrate mammalian cells. Associating cargo with these proteins, suchas plasmid DNA, RNA, or other proteins, can enable the functionaldelivery of these macromolecules into mammalian cells both in vitro andin vivo. The creation and characterization of supercharged proteins hasbeen reported in 2007 (Lawrence et al., 2007, Journal of the AmericanChemical Society 129, 10110-10112).

The nonviral delivery of RNA and plasmid DNA into mammalian cells arevaluable both for research and therapeutic applications (Akinc et al.,2010, Nat. Biotech. 26, 561-569). Purified +36 GFP protein (or othersuperpositively charged protein) is mixed with RNAs in the appropriateserum-free media and allowed to complex prior addition to cells.Inclusion of serum at this stage inhibits formation of the superchargedprotein-RNA complexes and reduces the effectiveness of the treatment.The following protocol has been found to be effective for a variety ofcell lines (McNaughton et al., 2009, Proc. Natl. Acad. Sci. USA 106,6111-6116) (However, pilot experiments varying the dose of protein andRNA should be performed to optimize the procedure for specific celllines): (1) One day before treatment, plate 1×105 cells per well in a48-well plate. (2) On the day of treatment, dilute purified +36 GFPprotein in serumfree media to a final concentration 200 nM. Add RNA to afinal concentration of 50 nM. Vortex to mix and incubate at roomtemperature for 10 min. (3) During incubation, aspirate media from cellsand wash once with PBS. (4) Following incubation of +36 GFP and RNA, addthe protein-RNA complexes to cells. (5) Incubate cells with complexes at37° C. for 4 h. (6) Following incubation, aspirate the media and washthree times with 20 U/mL heparin PBS. Incubate cells withserum-containing media for a further 48 h or longer depending upon theassay for activity. (7) Analyze cells by immunoblot, qPCR, phenotypicassay, or other appropriate method.

It has been further found +36 GFP to be an effective plasmid deliveryreagent in a range of cells. As plasmid DNA is a larger cargo thansiRNA, proportionately more +36 GFP protein is required to effectivelycomplex plasmids. For effective plasmid delivery Applicants havedeveloped a variant of +36 GFP bearing a C-terminal HA2 peptide tag, aknown endosome-disrupting peptide derived from the influenza virushemagglutinin protein. The following protocol has been effective in avariety of cells, but as above it is advised that plasmid DNA andsupercharged protein doses be optimized for specific cell lines anddelivery applications: (1) One day before treatment, plate 1×105 perwell in a 48-well plate. (2) On the day of treatment, dilute purified

36 GFP protein in serumfree media to a final concentration 2 mM. Add 1mg of plasmid DNA. Vortex to mix and incubate at room temperature for 10min. (3) During incubation, aspirate media from cells and wash once withPBS. (4) Following incubation of

36 GFP and plasmid DNA, gently add the protein-DNA complexes to cells.(5) Incubate cells with complexes at 37 C for 4 h. (6) Followingincubation, aspirate the media and wash with PBS. Incubate cells inserum-containing media and incubate for a further 24-48 h. (7) Analyzeplasmid delivery (e.g., by plasmid-driven gene expression) asappropriate.

See also, e.g., McNaughton et al., Proc. Natl. Acad. Sci. USA 106,6111-6116 (2009); Cronican et al., ACS Chemical Biology 5, 747-752(2010); Cronican et al., Chemistry & Biology 18, 833-838 (2011);Thompson et al., Methods in Enzymology 503, 293-319 (2012); Thompson, D.B., et al., Chemistry & Biology 19 (7), 831-843 (2012). The methods ofthe super charged proteins may be used and/or adapted for delivery ofthe AD-functionalized CRISPR Cas system of the present invention. Thesesystems in conjunction with herein teaching can be employed in thedelivery of AD-functionalized CRISPR Cas system(s) or component(s)thereof or nucleic acid molecule(s) coding therefor

Cell Penetrating Peptides (CPPs)

In yet another embodiment, cell penetrating peptides (CPPs) arecontemplated for the delivery of the AD-functionalized CRISPR Cassystem. CPPs are short peptides that facilitate cellular uptake ofvarious molecular cargo (from nanosize particles to small chemicalmolecules and large fragments of DNA). The term “cargo” as used hereinincludes but is not limited to the group consisting of therapeuticagents, diagnostic probes, peptides, nucleic acids, antisenseoligonucleotides, plasmids, proteins, particles, includingnanoparticles, liposomes, chromophores, small molecules and radioactivematerials. In aspects of the invention, the cargo may also comprise anycomponent of the AD-functionalized CRISPR Cas system or the entireAD-functionalized functional CRISPR Cas system. Aspects of the presentinvention further provide methods for delivering a desired cargo into asubject comprising: (a) preparing a complex comprising the cellpenetrating peptide of the present invention and a desired cargo, and(b) orally, intraarticularly, intraperitoneally, intrathecally,intrarterially, intranasally, intraparenchymally, subcutaneously,intramuscularly, intravenously, dermally, intrarectally, or topicallyadministering the complex to a subject. The cargo is associated with thepeptides either through chemical linkage via covalent bonds or throughnon-covalent interactions.

The function of the CPPs are to deliver the cargo into cells, a processthat commonly occurs through endocytosis with the cargo delivered to theendosomes of living mammalian cells. Cell-penetrating peptides are ofdifferent sizes, amino acid sequences, and charges but all CPPs have onedistinct characteristic, which is the ability to translocate the plasmamembrane and facilitate the delivery of various molecular cargoes to thecytoplasm or an organelle. CPP translocation may be classified intothree main entry mechanisms: direct penetration in the membrane,endocytosis-mediated entry, and translocation through the formation of atransitory structure. CPPs have found numerous applications in medicineas drug delivery agents in the treatment of different diseases includingcancer and virus inhibitors, as well as contrast agents for celllabeling. Examples of the latter include acting as a carrier for GFP, MMcontrast agents, or quantum dots. CPPs hold great potential as in vitroand in vivo delivery vectors for use in research and medicine. CPPstypically have an amino acid composition that either contains a highrelative abundance of positively charged amino acids such as lysine orarginine or has sequences that contain an alternating pattern ofpolar/charged amino acids and non-polar, hydrophobic amino acids. Thesetwo types of structures are referred to as polycationic or amphipathic,respectively. A third class of CPPs are the hydrophobic peptides,containing only apolar residues, with low net charge or have hydrophobicamino acid groups that are crucial for cellular uptake. One of theinitial CPPs discovered was the trans-activating transcriptionalactivator (Tat) from Human Immunodeficiency Virus 1 (HIV-1) which wasfound to be efficiently taken up from the surrounding media by numerouscell types in culture. Since then, the number of known CPPs has expandedconsiderably and small molecule synthetic analogues with more effectiveprotein transduction properties have been generated. CPPs include butare not limited to Penetratin, Tat (48-60), Transportan, and (R-AhX-R4)(Ahx=aminohexanoyl).

U.S. Pat. No. 8,372,951, provides a CPP derived from eosinophil cationicprotein (ECP) which exhibits highly cell-penetrating efficiency and lowtoxicity. Aspects of delivering the CPP with its cargo into a vertebratesubject are also provided. Further aspects of CPPs and their deliveryare described in U.S. Pat. Nos. 8,575,305; 8; 614,194 and 8,044,019.CPPs can be used to deliver the AD-functionalized CRISPR-Cas system orcomponents thereof. That CPPs can be employed to deliver theAD-functionalized CRISPR-Cas system or components thereof is alsoprovided in the manuscript “Gene disruption by cell-penetratingpeptide-mediated delivery of Cas9 protein and guide RNA”, by SureshRamakrishna, Abu-Bonsrah Kwaku Dad, Jagadish Beloor, et al. Genome Res.2014 Apr. 2, incorporated by reference in its entirety, wherein it isdemonstrated that treatment with CPP-conjugated recombinant Cas9 proteinand CPP-complexed guide RNAs lead to endogenous gene disruptions inhuman cell lines. In the paper the Cas9 protein was conjugated to CPPvia a thioether bond, whereas the guide RNA was complexed with CPP,forming condensed, positively charged particles. It was shown thatsimultaneous and sequential treatment of human cells, includingembryonic stem cells, dermal fibroblasts, HEK293T cells, HeLa cells, andembryonic carcinoma cells, with the modified Cas9 and guide RNA led toefficient gene disruptions with reduced off-target mutations relative toplasmid transfections.

Aerosol Delivery

Subjects treated for a lung disease may for example receivepharmaceutically effective amount of aerosolized AAV vector system perlung endobronchially delivered while spontaneously breathing. As such,aerosolized delivery is preferred for AAV delivery in general. Anadenovirus or an AAV particle may be used for delivery. Suitable geneconstructs, each operably linked to one or more regulatory sequences,may be cloned into the delivery vector.

Packaging and Promoters

The promoter used to drive CRISPR-Cas protein and adenosine deaminasecoding nucleic acid molecule expression can include AAV ITR, which canserve as a promoter. This is advantageous for eliminating the need foran additional promoter element (which can take up space in the vector).The additional space freed up can be used to drive the expression ofadditional elements (gRNA, etc.). Also, ITR activity is relativelyweaker, so can be used to reduce potential toxicity due to overexpression of Cas13.

For ubiquitous expression, promoters that can be used include: CMV, CAG,CBh, PGK, SV40, Ferritin heavy or light chains, etc. For brain or otherCNS expression, Synapsinl can be used for all neurons, CaMKIIalpha canbe used for excitatory neurons, GAD67 or GAD65 or VGAT can be used forGABAergic neurons. For liver expression, Albumin promoter can be used.For lung expression, SP-B can be used. For endothelial cells, ICAM canbe used. For hematopoietic cells, IFNbeta or CD45 can be used. ForOsteoblasts, the OG-2 can be used.

The promoter used to drive guide RNA can include Pol III promoters suchas U6 or H1, as well as use of Pol II promoter and intronic cassettes toexpress guide RNA.

Adeno Associated Virus (AAV)

The CRISPR-Cas protein, adenosine deaminase, and one or more guide RNAcan be delivered using adeno associated virus (AAV), lentivirus,adenovirus or other plasmid or viral vector types, in particular, usingformulations and doses from, for example, U.S. Pat. No. 8,454,972(formulations, doses for adenovirus), 8,404,658 (formulations, doses forAAV) and U.S. Pat. No. 5,846,946 (formulations, doses for DNA plasmids)and from clinical trials and publications regarding the clinical trialsinvolving lentivirus, AAV and adenovirus. For examples, for AAV, theroute of administration, formulation and dose can be as in U.S. Pat. No.8,454,972 and as in clinical trials involving AAV. For Adenovirus, theroute of administration, formulation and dose can be as in U.S. Pat. No.8,404,658 and as in clinical trials involving adenovirus. For plasmiddelivery, the route of administration, formulation and dose can be as inU.S. Pat. No 5,846,946 and as in clinical studies involving plasmids.Doses may be based on or extrapolated to an average 70 kg individual(e.g. a male adult human), and can be adjusted for patients, subjects,mammals of different weight and species. Frequency of administration iswithin the ambit of the medical or veterinary practitioner (e.g.,physician, veterinarian), depending on usual factors including the age,sex, general health, other conditions of the patient or subject and theparticular condition or symptoms being addressed. The viral vectors canbe injected into the tissue of interest. For cell-type specific genomemodification, the expression of Cas13 and adenosine deaminase can bedriven by a cell-type specific promoter. For example, liver-specificexpression might use the Albumin promoter and neuron-specific expression(e.g. for targeting CNS disorders) might use the Synapsin I promoter.

In terms of in vivo delivery, AAV is advantageous over other viralvectors for a couple of reasons: low toxicity (this may be due to thepurification method not requiring ultra centrifugation of cell particlesthat can activate the immune response); and low probability of causinginsertional mutagenesis because it doesn't integrate into the hostgenome.

AAV has a packaging limit of 4.5 or 4.75 Kb. This means that Cas13 aswell as a promoter and transcription terminator have to be all fit intothe same viral vector. Constructs larger than 4.5 or 4.75 Kb will leadto significantly reduced virus production. SpCas9 is quite large, thegene itself is over 4.1 Kb, which makes it difficult for packing intoAAV. Therefore embodiments of the invention include utilizing homologsof Cas13 that are shorter.

As to AAV, the AAV can be AAV1, AAV2, AAV5 or any combination thereof.One can select the AAV of the AAV with regard to the cells to betargeted; e.g., one can select AAV serotypes 1, 2, 5 or a hybrid capsidAAV1, AAV2, AAV5 or any combination thereof for targeting brain orneuronal cells; and one can select AAV4 for targeting cardiac tissue.AAV8 is useful for delivery to the liver. The herein promoters andvectors are preferred individually. A tabulation of certain AAVserotypes as to these cells (see Grimm, D. et al, J. Virol. 82:5887-5911 (2008)) is as follows:

TABLE 6 Cell Line AAV-1 AAV-2 AAV-3 AAV-4 AAV-5 AAV-6 AAV-8 AAV-9 Huh-713 100 2.5 0.0 0.1 10 0.7 0.0 HEK293 25 100 2.5 0.1 0.1 5 0.7 0.1 HeLa 3100 2.0 0.1 6.7 1 0.2 0.1 HepG2 3 100 16.7 0.3 1.7 5 0.3 ND Hep1A 20 1000.2 1.0 0.1 1 0.2 0.0 911 17 100 11 0.2 0.1 17 0.1 ND CHO 100 100 14 1.4333 50 10 1.0 COS 33 100 33 3.3 5.0 14 2.0 0.5 MeWo 10 100 20 0.3 6.7 101.0 0.2 NIH3T3 10 100 2.9 2.9 0.3 10 0.3 ND A549 14 100 20 ND 0.5 10 0.50.1 HT1180 20 100 10 0.1 0.3 33 0.5 0.1 Monocytes 1111 100 ND ND 1251429 ND ND Immature DC 2500 100 ND ND 222 2857 ND ND Mature DC 2222 100ND ND 333 3333 ND ND

Lentiviruses

Lentiviruses are complex retroviruses that have the ability to infectand express their genes in both mitotic and post-mitotic cells. The mostcommonly known lentivirus is the human immunodeficiency virus (HIV),which uses the envelope glycoproteins of other viruses to target a broadrange of cell types.

Lentiviruses may be prepared as follows. After cloning pCasES10 (whichcontains a lentiviral transfer plasmid backbone), HEK293FT at lowpassage (p=5) were seeded in a T-75 flask to 50% confluence the daybefore transfection in DMEM with 10% fetal bovine serum and withoutantibiotics. After 20 hours, media was changed to OptiMEM (serum-free)media and transfection was done 4 hours later. Cells were transfectedwith 10 μg of lentiviral transfer plasmid (pCasES10) and the followingpackaging plasmids: 5 μg of pMD2.G (VSV-g pseudotype), and 7.5 ug ofpsPAX2 (gag/pol/rev/tat). Transfection was done in 4 mL OptiMEM with acationic lipid delivery agent (50 uL Lipofectamine 2000 and 100 ul Plusreagent). After 6 hours, the media was changed to antibiotic-free DMEMwith 10% fetal bovine serum. These methods use serum during cellculture, but serum-free methods are preferred.

Lentivirus may be purified as follows. Viral supernatants were harvestedafter 48 hours. Supernatants were first cleared of debris and filteredthrough a 0.45 um low protein binding (PVDF) filter. They were then spunin a ultracentrifuge for 2 hours at 24,000 rpm. Viral pellets wereresuspended in 50 ul of DMEM overnight at 4C. They were then aliquottedand immediately frozen at −80° C.

In another embodiment, minimal non-primate lentiviral vectors based onthe equine infectious anemia virus (EIAV) are also contemplated,especially for ocular gene therapy (see, e.g., Balagaan, J Gene Med2006; 8: 275-285). In another embodiment, RetinoStat®, an equineinfectious anemia virus-based lentiviral gene therapy vector thatexpresses angiostatic proteins endostatin and angiostatin that isdelivered via a subretinal injection for the treatment of the web formof age-related macular degeneration is also contemplated (see, e.g.,Binley et al., HUMAN GENE THERAPY 23:980-991 (September 2012)) and thisvector may be modified for the AD-functionalized CRISPR-Cas system ofthe present invention.

In another embodiment, self-inactivating lentiviral vectors with ansiRNA targeting a common exon shared by HIV tat/rev, anucleolar-localizing TAR decoy, and an anti-CCR5-specific hammerheadribozyme (see, e.g., DiGiusto et al. (2010) Sci Transl Med 2:36ra43) maybe used/and or adapted to the AD-functionalized CRISPR-Cas system of thepresent invention. A minimum of 2.5×106 CD34+ cells per kilogram patientweight may be collected and prestimulated for 16 to 20 hours in X-VIVO15 medium (Lonza) containing 2 μmol/L-glutamine, stem cell factor (100ng/ml), Flt-3 ligand (Flt-3L) (100 ng/ml), and thrombopoietin (10 ng/ml)(CellGenix) at a density of 2×106 cells/ml. Prestimulated cells may betransduced with lentiviral at a multiplicity of infection of 5 for 16 to24 hours in 75-cm2 tissue culture flasks coated with fibronectin (25mg/cm2) (RetroNectin,Takara Bio Inc.).

Lentiviral vectors have been disclosed as in the treatment forParkinson's Disease, see, e.g., US Patent Publication No. 20120295960and U.S. Pat. Nos. 7,303,910 and 7,351,585. Lentiviral vectors have alsobeen disclosed for the treatment of ocular diseases, see e.g., US PatentPublication Nos. 20060281180, 20090007284, US20110117189; US20090017543;US20070054961, US20100317109. Lentiviral vectors have also beendisclosed for delivery to the brain, see, e.g., US Patent PublicationNos. US20110293571; US20110293571, US20040013648, US20070025970,US20090111106 and U.S. Pat. No. 7,259,015.

Application in Non-Animal Organisms

The System(s) (e.g., single or multiplexed) can be used in conjunctionwith recent advances in crop genomics. The systems described herein canbe used to perform efficient and cost effective plant gene or genomeinterrogation or editing or manipulation—for instance, for rapidinvestigation and/or selection and/or interrogations and/or comparisonand/or manipulations and/or transformation of plant genes or genomes;e.g., to create, identify, develop, optimize, or confer trait(s) orcharacteristic(s) to plant(s) or to transform a plant genome. There canaccordingly be improved production of plants, new plants with newcombinations of traits or characteristics or new plants with enhancedtraits. The System can be used with regard to plants in Site-DirectedIntegration (SDI) or Gene Editing (GE) or any Near Reverse Breeding(NRB) or Reverse Breeding (RB) techniques. Aspects of utilizing theherein described Cas13 effector protein systems may be analogous to theuse of the CRISPR-Cas (e.g. CRISPR-Cas9) system in plants, and mentionis made of the University of Arizona web site “CRISPR-PLANT”(www.genome.arizona.edu/crispr/) (supported by Penn State and AGI).Embodiments of the invention can be used in genome editing in plants orwhere RNAi or similar genome editing techniques have been usedpreviously; see, e.g., Nekrasov, “Plant genome editing made easy:targeted mutagenesis in model and crop plants using the CRISPR-Cassystem,” Plant Methods 2013, 9:39 (doi:10.1186/1746-4811-9-39); Brooks,“Efficient gene editing in tomato in the first generation using theCRISPR-Cas9 system,” Plant Physiology September 2014 pp 114.247577;Shan, “Targeted genome modification of crop plants using a CRISPR-Cassystem,” Nature Biotechnology 31, 686-688 (2013); Feng, “Efficientgenome editing in plants using a CRISPR-Cas system,” Cell Research(2013) 23:1229-1232. doi:10.1038/cr.2013.114; published online 20 Aug.2013; Xie, “RNA-guided genome editing in plants using a CRISPR-Cassystem,” Mol Plant. 2013 November; 6(6):1975-83. doi: 10.1093/mp/sst119.Epub 2013 Aug. 17; Xu, “Gene targeting using the Agrobacteriumtumefaciens-mediated CRISPR-Cas system in rice,” Rice 2014, 7:5 (2014),Zhou et al., “Exploiting SNPs for biallelic CRISPR mutations in theoutcrossing woody perennial Populus reveals 4-coumarate: CoA ligasespecificity and Redundancy,” New Phytologist (2015) (Forum) 1-4(available online only at www.newphytologist.com); Caliando et al,“Targeted DNA degradation using a CRISPR device stably carried in thehost genome, NATURE COMMUNICATIONS 6:6989, DOI: 10.1038/ncomms7989,www.nature.com/naturecommunications DOI: 10.1038/ncomms7989; U.S. Pat.No. 6,603,061—Agrobacterium-Mediated Plant Transformation Method; U.S.Pat. No. 7,868,149—Plant Genome Sequences and Uses Thereof and US2009/0100536—Transgenic Plants with Enhanced Agronomic Traits, all thecontents and disclosure of each of which are herein incorporated byreference in their entirety. In the practice of the invention, thecontents and disclosure of Morrell et al “Crop genomics: advances andapplications,” Nat Rev Genet. 2011 Dec. 29; 13(2):85-96; each of whichis incorporated by reference herein including as to how hereinembodiments may be used as to plants. Accordingly, reference herein toanimal cells may also apply, mutatis mutandis, to plant cells unlessotherwise apparent; and, the enzymes herein having reduced off-targeteffects and systems employing such enzymes can be used in plantapplications, including those mentioned herein.

Application of Systems to Plants and Yeast

In general, the term “plant” relates to any various photosynthetic,eukaryotic, unicellular or multicellular organism of the kingdom Plantaecharacteristically growing by cell division, containing chloroplasts,and having cell walls comprised of cellulose. The term plant encompassesmonocotyledonous and dicotyledonous plants. Specifically, the plants areintended to comprise without limitation angiosperm and gymnosperm plantssuch as acacia, alfalfa, amaranth, apple, apricot, artichoke, ash tree,asparagus, avocado, banana, barley, beans, beet, birch, beech,blackberry, blueberry, broccoli, Brussel's sprouts, cabbage, canola,cantaloupe, carrot, cassava, cauliflower, cedar, a cereal, celery,chestnut, cherry, Chinese cabbage, citrus, clementine, clover, coffee,corn, cotton, cowpea, cucumber, cypress, eggplant, elm, endive,eucalyptus, fennel, figs, fir, geranium, grape, grapefruit, groundnuts,ground cherry, gum hemlock, hickory, kale, kiwifruit, kohlrabi, larch,lettuce, leek, lemon, lime, locust, pine, maidenhair, maize, mango,maple, melon, millet, mushroom, mustard, nuts, oak, oats, oil palm,okra, onion, orange, an ornamental plant or flower or tree, papaya,palm, parsley, parsnip, pea, peach, peanut, pear, peat, pepper,persimmon, pigeon pea, pine, pineapple, plantain, plum, pomegranate,potato, pumpkin, radicchio, radish, rapeseed, raspberry, rice, rye,sorghum, safflower, sallow, soybean, spinach, spruce, squash,strawberry, sugar beet, sugarcane, sunflower, sweet potato, sweet corn,tangerine, tea, tobacco, tomato, trees, triticale, turf grasses,turnips, vine, walnut, watercress, watermelon, wheat, yams, yew, andzucchini. The term plant also encompasses Algae, which are mainlyphotoautotrophs unified primarily by their lack of roots, leaves andother organs that characterize higher plants.

The methods for genome editing using the System as described herein canbe used to confer desired traits on essentially any plant. A widevariety of plants and plant cell systems may be engineered for thedesired physiological and agronomic characteristics described hereinusing the nucleic acid constructs of the present disclosure and thevarious transformation methods mentioned above. In preferredembodiments, target plants and plant cells for engineering include, butare not limited to, those monocotyledonous and dicotyledonous plants,such as crops including grain crops (e.g., wheat, maize, rice, millet,barley), fruit crops (e.g., tomato, apple, pear, strawberry, orange),forage crops (e.g., alfalfa), root vegetable crops (e.g., carrot,potato, sugar beets, yam), leafy vegetable crops (e.g., lettuce,spinach); flowering plants (e.g., petunia, rose, chrysanthemum),conifers and pine trees (e.g., pine fir, spruce); plants used inphytoremediation (e.g., heavy metal accumulating plants); oil crops(e.g., sunflower, rape seed) and plants used for experimental purposes(e.g., Arabidopsis). Thus, the methods and systems can be used over abroad range of plants, such as for example with dicotyledonous plantsbelonging to the orders Magniolales, Illiciales, Laurales, Piperales,Aristochiales, Nymphaeales, Ranunculales, Papeverales, Sarraceniaceae,Trochodendrales, Hamamelidales, Eucomiales, Leitneriales, Myricales,Fagales, Casuarinales, Caryophyllales, Batales, Polygonales,Plumbaginales, Dilleniales, Theales, Malvales, Urticales, Lecythidales,Violales, Salicales, Capparales, Ericales, Diapensales, Ebenales,Primulales, Rosales, Fabales, Podostemales, Haloragales, Myrtales,Cornales, Proteales, San tales, Rafflesiales, Celastrales, Euphorbiales,Rhamnales, Sapindales, Juglandales, Geraniales, Polygalales, Umbellales,Gentianales, Polemoniales, Lamiales, Plantaginales, Scrophulariales,Campanulales, Rubiales, Dipsacales, and Asterales; the methods andCRISPR-Cas systems can be used with monocotyledonous plants such asthose belonging to the orders Alismatales, Hydrocharitales, Najadales,Triuridales, Commelinales, Eriocaulales, Restionales, Poales, Juncales,Cyperales, Typhales, Bromeliales, Zingiberales, Arecales, Cyclanthales,Pandanales, Arales, Lilliales, and Orchid ales, or with plants belongingto Gymnospermae, e.g those belonging to the orders Pinales, Ginkgoales,Cycadales, Araucariales, Cupressales and Gnetales.

The Systems and methods of use described herein can be used over a broadrange of plant species, included in the non-limitative list of dicot,monocot or gymnosperm genera hereunder: Atropa, Alseodaphne, Anacardium,Arachis, Beilschmiedia, Brassica, Carthamus, Cocculus, Croton, Cucumis,Citrus, Citrullus, Capsicum, Catharanthus, Cocos, Coffea, Cucurbita,Daucus, Duguetia, Eschscholzia, Ficus, Fragaria, Glaucium, Glycine,Gossypium, Helianthus, Hevea, Hyoscyamus, Lactuca, Landolphia, Linum,Litsea, Lycopersicon, Lupinus, Manihot, Majorana, Malus, Medicago,Nicotiana, Olea, Parthenium, Papaver, Persea, Phaseolus, Pistacia,Pisum, Pyrus, Prunus, Raphanus, Ricinus, Senecio, Sinomenium, Stephania,Sinapis, Solanum, Theobroma, Trifolium, Trigonella, Vicia, Vinca, Vilis,and Vigna; and the genera Allium, Andropogon, Aragrostis, Asparagus,Avena, Cynodon, Elaeis, Festuca, Festulolium, Heterocallis, Hordeum,Lemna, Lolium, Musa, Oryza, Panicum, Pannesetum, Phleum, Poa, Secale,Sorghum, Triticum, Zea, Abies, Cunninghamia, Ephedra, Picea, Pinus, andPseudotsuga.

The Systems and methods of use can also be used over a broad range of“algae” or “algae cells”; including for example algea selected fromseveral eukaryotic phyla, including the Rhodophyta (red algae),Chlorophyta (green algae), Phaeophyta (brown algae), Bacillariophyta(diatoms), Eustigmatophyta and dinoflagellates as well as theprokaryotic phylum Cyanobacteria (blue-green algae). The term “algae”includes for example algae selected from: Amphora, Anabaena,Anikstrodesmis, Botryococcus, Chaetoceros, Chlamydomonas, Chlorella,Chlorococcum, Cyclotella, Cylindrotheca, Dunaliella, Emiliana, Euglena,Hematococcus, Isochrysis, Monochrysis, Monoraphidium, Nannochloris,Nannnochloropsis, Navicula, Nephrochloris, Nephroselmis, Nitzschia,Nodularia, Nostoc, Oochromonas, Oocystis, Oscillartoria, Pavlova,Phaeodactylum, Playtmonas, Pleurochrysis, Porhyra, Pseudoanabaena,Pyramimonas, Stichococcus, Synechococcus, Synechocystis, Tetraselmis,Thalassiosira, and Trichodesmium.

A part of a plant, i.e., a “plant tissue” may be treated according tothe methods of the present invention to produce an improved plant. Planttissue also encompasses plant cells. The term “plant cell” as usedherein refers to individual units of a living plant, either in an intactwhole plant or in an isolated form grown in in vitro tissue cultures, onmedia or agar, in suspension in a growth media or buffer or as a part ofhigher organized unites, such as, for example, plant tissue, a plantorgan, or a whole plant.

A “protoplast” refers to a plant cell that has had its protective cellwall completely or partially removed using, for example, mechanical orenzymatic means resulting in an intact biochemical competent unit ofliving plant that can reform their cell wall, proliferate and regenerategrow into a whole plant under proper growing conditions.

The term “transformation” broadly refers to the process by which a planthost is genetically modified by the introduction of DNA by means ofAgrobacteria or one of a variety of chemical or physical methods. Asused herein, the term “plant host” refers to plants, including anycells, tissues, organs, or progeny of the plants. Many suitable planttissues or plant cells can be transformed and include, but are notlimited to, protoplasts, somatic embryos, pollen, leaves, seedlings,stems, calli, stolons, microtubers, and shoots. A plant tissue alsorefers to any clone of such a plant, seed, progeny, propagule whethergenerated sexually or asexually, and descendents of any of these, suchas cuttings or seed.

The term “transformed” as used herein, refers to a cell, tissue, organ,or organism into which a foreign DNA molecule, such as a construct, hasbeen introduced. The introduced DNA molecule may be integrated into thegenomic DNA of the recipient cell, tissue, organ, or organism such thatthe introduced DNA molecule is transmitted to the subsequent progeny. Inthese embodiments, the “transformed” or “transgenic” cell or plant mayalso include progeny of the cell or plant and progeny produced from abreeding program employing such a transformed plant as a parent in across and exhibiting an altered phenotype resulting from the presence ofthe introduced DNA molecule. Preferably, the transgenic plant is fertileand capable of transmitting the introduced DNA to progeny through sexualreproduction.

The term “progeny”, such as the progeny of a transgenic plant, is onethat is born of, begotten by, or derived from a plant or the transgenicplant. The introduced DNA molecule may also be transiently introducedinto the recipient cell such that the introduced DNA molecule is notinherited by subsequent progeny and thus not considered “transgenic”.Accordingly, as used herein, a “non-transgenic” plant or plant cell is aplant which does not contain a foreign DNA stably integrated into itsgenome.

The term “plant promoter” as used herein is a promoter capable ofinitiating transcription in plant cells, whether or not its origin is aplant cell. Exemplary suitable plant promoters include, but are notlimited to, those that are obtained from plants, plant viruses, andbacteria such as Agrobacterium or Rhizobium which comprise genesexpressed in plant cells.

As used herein, a “fungal cell” refers to any type of eukaryotic cellwithin the kingdom of fungi. Phyla within the kingdom of fungi includeAscomycota, Basidiomycota, Blastocladiomycota, Chytridiomycota,Glomeromycota, Microsporidia, and Neocallimastigomycota. Fungal cellsmay include yeasts, molds, and filamentous fungi. In some embodiments,the fungal cell is a yeast cell.

As used herein, the term “yeast cell” refers to any fungal cell withinthe phyla Ascomycota and Basidiomycota. Yeast cells may include buddingyeast cells, fission yeast cells, and mold cells. Without being limitedto these organisms, many types of yeast used in laboratory andindustrial settings are part of the phylum Ascomycota. In someembodiments, the yeast cell is an S. cerervisiae, Kluyveromycesmarxianus, or Issatchenkia orientalis cell. Other yeast cells mayinclude without limitation Candida spp. (e.g., Candida albicans),Yarrowia spp. (e.g., Yarrowia lipolytica), Pichia spp. (e.g., Pichiapastoris), Kluyveromyces spp. (e.g., Kluyveromyces lactis andKluyveromyces marxianus), Neurospora spp. (e.g., Neurospora crassa),Fusarium spp. (e.g., Fusarium oxysporum), and Issatchenkia spp. (e.g.,Issatchenkia orientalis, a.k.a. Pichia kudriavzevii and Candidaacidothermophilum). In some embodiments, the fungal cell is afilamentous fungal cell. As used herein, the term “filamentous fungalcell” refers to any type of fungal cell that grows in filaments, i.e.,hyphae or mycelia. Examples of filamentous fungal cells may includewithout limitation Aspergillus spp. (e.g., Aspergillus niger),Trichoderma spp. (e.g., Trichoderma reesei), Rhizopus spp. (e.g.,Rhizopus oryzae), and Mortierella spp. (e.g., Mortierella isabellina).

In some embodiments, the fungal cell is an industrial strain. As usedherein, “industrial strain” refers to any strain of fungal cell used inor isolated from an industrial process, e.g., production of a product ona commercial or industrial scale. Industrial strain may refer to afungal species that is typically used in an industrial process, or itmay refer to an isolate of a fungal species that may be also used fornon-industrial purposes (e.g., laboratory research). Examples ofindustrial processes may include fermentation (e.g., in production offood or beverage products), distillation, biofuel production, productionof a compound, and production of a polypeptide. Examples of industrialstrains may include, without limitation, JAY270 and ATCC4124.

In some embodiments, the fungal cell is a polyploid cell. As usedherein, a “polyploid” cell may refer to any cell whose genome is presentin more than one copy. A polyploid cell may refer to a type of cell thatis naturally found in a polyploid state, or it may refer to a cell thathas been induced to exist in a polyploid state (e.g., through specificregulation, alteration, inactivation, activation, or modification ofmeiosis, cytokinesis, or DNA replication). A polyploid cell may refer toa cell whose entire genome is polyploid, or it may refer to a cell thatis polyploid in a particular genomic locus of interest. Without wishingto be bound to theory, it is thought that the abundance of guideRNA maymore often be a rate-limiting component in genome engineering ofpolyploid cells than in haploid cells, and thus the methods using theSystem described herein may take advantage of using a certain fungalcell type.

In some embodiments, the fungal cell is a diploid cell. As used herein,a “diploid” cell may refer to any cell whose genome is present in twocopies. A diploid cell may refer to a type of cell that is naturallyfound in a diploid state, or it may refer to a cell that has beeninduced to exist in a diploid state (e.g., through specific regulation,alteration, inactivation, activation, or modification of meiosis,cytokinesis, or DNA replication). For example, the S. cerevisiae strainS228C may be maintained in a haploid or diploid state. A diploid cellmay refer to a cell whose entire genome is diploid, or it may refer to acell that is diploid in a particular genomic locus of interest. In someembodiments, the fungal cell is a haploid cell. As used herein, a“haploid” cell may refer to any cell whose genome is present in onecopy. A haploid cell may refer to a type of cell that is naturally foundin a haploid state, or it may refer to a cell that has been induced toexist in a haploid state (e.g., through specific regulation, alteration,inactivation, activation, or modification of meiosis, cytokinesis, orDNA replication). For example, the S. cerevisiae strain S228C may bemaintained in a haploid or diploid state. A haploid cell may refer to acell whose entire genome is haploid, or it may refer to a cell that ishaploid in a particular genomic locus of interest.

As used herein, a “yeast expression vector” refers to a nucleic acidthat contains one or more sequences encoding an RNA and/or polypeptideand may further contain any desired elements that control the expressionof the nucleic acid(s), as well as any elements that enable thereplication and maintenance of the expression vector inside the yeastcell. Many suitable yeast expression vectors and features thereof areknown in the art; for example, various vectors and techniques areillustrated in in Yeast Protocols, 2nd edition, Xiao, W., ed. (HumanaPress, New York, 2007) and Buckholz, R. G. and Gleeson, M. A. (1991)Biotechnology (NY) 9(11): 1067-72. Yeast vectors may contain, withoutlimitation, a centromeric (CEN) sequence, an autonomous replicationsequence (ARS), a promoter, such as an RNA Polymerase III promoter,operably linked to a sequence or gene of interest, a terminator such asan RNA polymerase III terminator, an origin of replication, and a markergene (e.g., auxotrophic, antibiotic, or other selectable markers).Examples of expression vectors for use in yeast may include plasmids,yeast artificial chromosomes, 2μ plasmids, yeast integrative plasmids,yeast replicative plasmids, shuttle vectors, and episomal plasmids.

Stable Integration of System Components in the Genome of Plants andPlant Cells

In particular embodiments, it is envisaged that the polynucleotidesencoding the components of the System are introduced for stableintegration into the genome of a plant cell. In these embodiments, thedesign of the transformation vector or the expression system can beadjusted depending on for when, where and under what conditions theguide RNA and/or fusion protein of adenosine deaminase and Cas13 areexpressed.

In particular embodiments, it is envisaged to introduce the componentsof the System stably into the genomic DNA of a plant cell. Additionallyor alternatively, it is envisaged to introduce the components of theSystem for stable integration into the DNA of a plant organelle such as,but not limited to a plastid, e mitochondrion or a chloroplast.

The expression system for stable integration into the genome of a plantcell may contain one or more of the following elements: a promoterelement that can be used to express the RNA and/or fusion protein ofadenosine deaminase and Cas13 in a plant cell; a 5′ untranslated regionto enhance expression; an intron element to further enhance expressionin certain cells, such as monocot cells; a multiple-cloning site toprovide convenient restriction sites for inserting the guide RNA and/orthe fusion protein of adenosine deaminase and Cas13 encoding sequencesand other desired elements; and a 3′ untranslated region to provide forefficient termination of the expressed transcript.

The elements of the expression system may be on one or more expressionconstructs which are either circular such as a plasmid or transformationvector, or non-circular such as linear double stranded DNA.

In a particular embodiment, a AD-functionalized CRISPR expression systemcomprises at least: a nucleotide sequence encoding a guide RNA (gRNA)that hybridizes with a target sequence in a plant, and wherein the guideRNA comprises a guide sequence and a direct repeat sequence, and anucleotide sequence encoding a fusion protein of adenosine deaminase andCas13, wherein components (a) or (b) are located on the same or ondifferent constructs, and whereby the different nucleotide sequences canbe under control of the same or a different regulatory element operablein a plant cell.

DNA construct(s) containing the components of the System, and, whereapplicable, template sequence may be introduced into the genome of aplant, plant part, or plant cell by a variety of conventionaltechniques. The process generally comprises the steps of selecting asuitable host cell or host tissue, introducing the construct(s) into thehost cell or host tissue, and regenerating plant cells or plantstherefrom.

In particular embodiments, the DNA construct may be introduced into theplant cell using techniques such as but not limited to electroporation,microinjection, aerosol beam injection of plant cell protoplasts, or theDNA constructs can be introduced directly to plant tissue usingbiolistic methods, such as DNA particle bombardment (see also Fu et al.,Transgenic Res. 2000 February; 9(1):11-9). The basis of particlebombardment is the acceleration of particles coated with gene/s ofinterest toward cells, resulting in the penetration of the protoplasm bythe particles and typically stable integration into the genome. (seee.g. Klein et al, Nature (1987), Klein et ah, Bio/Technology (1992),Casas et ah, Proc. Natl. Acad. Sci. USA (1993).).

In particular embodiments, the DNA constructs containing components ofthe System may be introduced into the plant by Agrobacterium-mediatedtransformation. The DNA constructs may be combined with suitable T-DNAflanking regions and introduced into a conventional Agrobacteriumtumefaciens host vector. The foreign DNA can be incorporated into thegenome of plants by infecting the plants or by incubating plantprotoplasts with Agrobacterium bacteria, containing one or more Ti(tumor-inducing) plasmids. (see e.g. Fraley et al., (1985), Rogers etal., (1987) and U.S. Pat. No. 5,563,055).

Plant Promoters

In order to ensure appropriate expression in a plant cell, thecomponents of the System described herein are typically placed undercontrol of a plant promoter, i.e. a promoter operable in plant cells.The use of different types of promoters is envisaged.

A constitutive plant promoter is a promoter that is able to express theopen reading frame (ORF) that it controls in all or nearly all of theplant tissues during all or nearly all developmental stages of the plant(referred to as “constitutive expression”). One non-limiting example ofa constitutive promoter is the cauliflower mosaic virus 35S promoter.“Regulated promoter” refers to promoters that direct gene expression notconstitutively, but in a temporally- and/or spatially-regulated manner,and includes tissue-specific, tissue-preferred and inducible promoters.Different promoters may direct the expression of a gene in differenttissues or cell types, or at different stages of development, or inresponse to different environmental conditions. In particularembodiments, one or more of the AD-functionalized CRISPR components areexpressed under the control of a constitutive promoter, such as thecauliflower mosaic virus 35S promoter issue-preferred promoters can beutilized to target enhanced expression in certain cell types within aparticular plant tissue, for instance vascular cells in leaves or rootsor in specific cells of the seed. Examples of particular promoters foruse in the System are found in Kawamata et al., (1997) Plant CellPhysiol 38:792-803; Yamamoto et al., (1997) Plant J 12:255-65; Hire etal, (1992) Plant Mol Biol 20:207-18, Kuster et al, (1995) Plant Mol Biol29:759-72, and Capana et al., (1994) Plant Mol Biol 25:681-91.

Inducible promoters can be of interest to express one or more of thecomponents of the System under limited circumstances to avoidnon-specific activity of the deaminase. In particular embodiments, oneor more elements of the System are expressed under control of aninducible promoter. Examples of promoters that are inducible and thatallow for spatiotemporal control of gene editing or gene expression mayuse a form of energy. The form of energy may include but is not limitedto sound energy, electromagnetic radiation, chemical energy and/orthermal energy. Examples of inducible systems include tetracyclineinducible promoters (Tet-On or Tet-Off), small molecule two-hybridtranscription activations systems (FKBP, ABA, etc), or light induciblesystems (Phytochrome, LOV domains, or cryptochrome)., such as a LightInducible Transcriptional Effector (LITE) that direct changes intranscriptional activity in a sequence-specific manner. The componentsof a light inducible system may include a fusion protein of adenosinedeaminase and Cas13, a light-responsive cytochrome heterodimer (e.g.from Arabidopsis thaliana). Further examples of inducible DNA bindingproteins and methods for their use are provided in U.S. 61/736465 andU.S. 61/721,283, which is hereby incorporated by reference in itsentirety.

In particular embodiments, transient or inducible expression can beachieved by using, for example, chemical-regulated promotors, i.e.whereby the application of an exogenous chemical induces geneexpression. Modulating of gene expression can also be obtained by achemical-repressible promoter, where application of the chemicalrepresses gene expression. Chemical-inducible promoters include, but arenot limited to, the maize ln2-2 promoter, activated by benzenesulfonamide herbicide safeners (De Veylder et al., (1997) Plant CellPhysiol 38:568-77), the maize GST promoter (GST-11-27, WO93/01294),activated by hydrophobic electrophilic compounds used as pre-emergentherbicides, and the tobacco PR-1 a promoter (Ono et al., (2004) BiosciBiotechnol Biochem 68:803-7) activated by salicylic acid. Promoterswhich are regulated by antibiotics, such as tetracycline-inducible andtetracycline-repressible promoters (Gatz et al., (1991) Mol Gen Genet227:229-37; U.S. Pat. Nos. 5,814,618 and 5,789,156) can also be usedherein.

Translocation to and/or Expression in Specific Plant Organelles

The expression system may comprise elements for translocation to and/orexpression in a specific plant organelle.

Chloroplast Targeting

In particular embodiments, it is envisaged that the System is used tospecifically modify chloroplast genes or to ensure expression in thechloroplast. For this purpose use is made of chloroplast transformationmethods or compartimentalization of the AD-functionalized CRISPRcomponents to the chloroplast. For instance, the introduction of geneticmodifications in the plastid genome can reduce biosafety issues such asgene flow through pollen.

Methods of chloroplast transformation are known in the art and includeParticle bombardment, PEG treatment, and microinjection. Additionally,methods involving the translocation of transformation cassettes from thenuclear genome to the pastid can be used as described in WO2010061186.

Alternatively, it is envisaged to target one or more of theAD-functionalized CRISPR components to the plant chloroplast. This isachieved by incorporating in the expression construct a sequenceencoding a chloroplast transit peptide (CTP) or plastid transit peptide,operably linked to the 5′ region of the sequence encoding the fusionprotein of adenosine deaminase and Cas13. The CTP is removed in aprocessing step during translocation into the chloroplast. Chloroplasttargeting of expressed proteins is well known to the skilled artisan(see for instance Protein Transport into Chloroplasts, 2010, AnnualReview of Plant Biology, Vol. 61: 157-180) . In such embodiments it isalso desired to target the guide RNA to the plant chloroplast. Methodsand constructs which can be used for translocating guide RNA into thechloroplast by means of a chloroplast localization sequence aredescribed, for instance, in US 20040142476, incorporated herein byreference. Such variations of constructs can be incorporated into theexpression systems of the invention to efficiently translocate theSystem components.

Introduction of Polynucleotides Encoding the System in Algae Cells.

Transgenic algae (or other plants such as rape) may be particularlyuseful in the production of vegetable oils or biofuels such as alcohols(especially methanol and ethanol) or other products. These may beengineered to express or overexpress high levels of oil or alcohols foruse in the oil or biofuel industries.

U.S. Pat. No. 8,945,839 describes a method for engineering Micro-Algae(Chlamydomonas reinhardtii cells) species) using Cas9. Using similartools, the methods of the System described herein can be applied onChlamydomonas species and other algae. In particular embodiments, aCRISPR-Cas protein (e.g., Cas13), adenosine deaminase (which may befused to the CRISPR-Cas protein or an aptamer-binding adaptor protein),and guide RNA are introduced in algae expressed using a vector thatexpresses the fusion protein of adenosine deaminase and Cas13 under thecontrol of a constitutive promoter such as Hsp70A-Rbc S2 orBeta2-tubulin. Guide RNA is optionally delivered using a vectorcontaining T7 promoter. Alternatively, Cas13 mRNA and in vitrotranscribed guide RNA can be delivered to algal cells. Electroporationprotocols are available to the skilled person such as the standardrecommended protocol from the GeneArt Chlamydomonas Engineering kit.

Introduction of System Components in Yeast Cells

In particular embodiments, the invention relates to the use of theSystem for genome editing of yeast cells. Methods for transforming yeastcells which can be used to introduce polynucleotides encoding the Systemcomponents are described in Kawai et al., 2010, Bioeng Bugs. 2010November-December; 1(6): 395-403). Non-limiting examples includetransformation of yeast cells by lithium acetate treatment (which mayfurther include carrier DNA and PEG treatment), bombardment or byelectroporation.

Transient Expression of System Components in Plants and Plant Cell

In particular embodiments, it is envisaged that the guide RNA and/orCRISPR-Cas gene are transiently expressed in the plant cell. In theseembodiments, the System can ensure modification of a target gene onlywhen both the guide RNA, the CRISPR-Cas protein (e.g., Cas13), andadenosine deaminase (which may be fused to the CRISPR-Cas protein or anaptamer-binding adaptor protein), are present in a cell, such thatgenomic modification can further be controlled. As the expression of theCRISPR-Cas protein is transient, plants regenerated from such plantcells typically contain no foreign DNA. In particular embodiments theCRISPR-Cas protein is stably expressed by the plant cell and the guidesequence is transiently expressed.

In particular embodiments, the System components can be introduced inthe plant cells using a plant viral vector (Scholthof et al. 1996, AnnuRev Phytopathol. 1996; 34:299-323). In further particular embodiments,said viral vector is a vector from a DNA virus. For example, geminivirus(e.g., cabbage leaf curl virus, bean yellow dwarf virus, wheat dwarfvirus, tomato leaf curl virus, maize streak virus, tobacco leaf curlvirus, or tomato golden mosaic virus) or nanovirus (e.g., Faba beannecrotic yellow virus). In other particular embodiments, said viralvector is a vector from an RNA virus. For example, tobravirus (e.g.,tobacco rattle virus, tobacco mosaic virus), potexvirus (e.g., potatovirus X), or hordeivirus (e.g., barley stripe mosaic virus). Thereplicating genomes of plant viruses are non-integrative vectors.

In particular embodiments, the vector used for transient expression ofSystem is for instance a pEAQ vector, which is tailored forAgrobacterium-mediated transient expression (Sainsbury F. et al., PlantBiotechnol J. 2009 September; 7(7):682-93) in the protoplast. Precisetargeting of genomic locations was demonstrated using a modified CabbageLeaf Curl virus (CaLCuV) vector to express guide RNAs in stabletransgenic plants expressing a CRISPR enzyme (Scientific Reports 5,Article number: 14926 (2015), doi:10.1038/srep14926).

In particular embodiments, double-stranded DNA fragments encoding theguide RNA and/or the CRISPR-Cas gene can be transiently introduced intothe plant cell. In such embodiments, the introduced double-stranded DNAfragments are provided in sufficient quantity to modify the cell but donot persist after a contemplated period of time has passed or after oneor more cell divisions. Methods for direct DNA transfer in plants areknown by the skilled artisan (see for instance Davey et al. Plant MolBiol. 1989 September; 13(3):273-85.)

In other embodiments, an RNA polynucleotide encoding the CRISPR-Casprotein (e.g., Cas13) and/or adenosine deaminase (which may be fused tothe CRISPR-Cas protein or an aptamer-binding adaptor protein) isintroduced into the plant cell, which is then translated and processedby the host cell generating the protein in sufficient quantity to modifythe cell (in the presence of at least one guide RNA) but which does notpersist after a contemplated period of time has passed or after one ormore cell divisions. Methods for introducing mRNA to plant protoplastsfor transient expression are known by the skilled artisan (see forinstance in Gallie, Plant Cell Reports (1993), 13; 119-122).

Combinations of the different methods described above are alsoenvisaged.

Delivery of System Components to the Plant Cell

In particular embodiments, it is of interest to deliver one or morecomponents of the System directly to the plant cell. This is ofinterest, inter alia, for the generation of non-transgenic plants (seebelow). In particular embodiments, one or more of the System componentsis prepared outside the plant or plant cell and delivered to the cell.For instance in particular embodiments, the CRISPR-Cas protein isprepared in vitro prior to introduction to the plant cell. TheCRISPR-Cas protein can be prepared by various methods known by one ofskill in the art and include recombinant production. After expression,the CRISPR-Cas protein is isolated, refolded if needed, purified andoptionally treated to remove any purification tags, such as a His-tag.Once crude, partially purified, or more completely purified CRISPR-Casprotein is obtained, the protein may be introduced to the plant cell.

In particular embodiments, the CRISPR-Cas protein is mixed with guideRNA targeting the gene of interest to form a pre-assembledribonucleoprotein.

The individual components or pre-assembled ribonucleoprotein can beintroduced into the plant cell via electroporation, by bombardment withCRISPR-Cas-associated gene product coated particles, by chemicaltransfection or by some other means of transport across a cell membrane.For instance, transfection of a plant protoplast with a pre-assembledCRISPR ribonucleoprotein has been demonstrated to ensure targetedmodification of the plant genome (as described by Woo et al. NatureBiotechnology, 2015; DOI: 10.1038/nbt.3389).

In particular embodiments, the System components are introduced into theplant cells using nanoparticles. The components, either as protein ornucleic acid or in a combination thereof, can be uploaded onto orpackaged in nanoparticles and applied to the plants (such as forinstance described in WO 2008042156 and US 20130185823). In particular,embodiments of the invention comprise nanoparticles uploaded with orpacked with DNA molecule(s) encoding the CRISPR-Cas protein (e.g.,Cas13), DNA molecule(s) encoding adenosine deaminase (which may be fusedto the CRISPR-Cas protein or an aptamer-binding adaptor protein), andDNA molecules encoding the guide RNA and/or isolated guide RNA asdescribed in WO2015089419.

Further means of introducing one or more components of the System to theplant cell is by using cell penetrating peptides (CPP). Accordingly, inparticular, embodiments the invention comprises compositions comprisinga cell penetrating peptide linked to the CRISPR-Cas protein. Inparticular embodiments of the present invention, the CRISPR-Cas proteinand/or guide RNA is coupled to one or more CPPs to effectively transportthem inside plant protoplasts. Ramakrishna (Genome Res. 2014 June;24(6):1020-7 for Cas9 in human cells). In other embodiments, theCRISPR-Cas gene and/or guide RNA are encoded by one or more circular ornon-circular DNA molecule(s) which are coupled to one or more CPPs forplant protoplast delivery. The plant protoplasts are then regenerated toplant cells and further to plants. CPPs are generally described as shortpeptides of fewer than 35 amino acids either derived from proteins orfrom chimeric sequences which are capable of transporting biomoleculesacross cell membrane in a receptor independent manner. CPP can becationic peptides, peptides having hydrophobic sequences, amphipaticpeptides, peptides having proline-rich and anti-microbial sequence, andchimeric or bipartite peptides (Pooga and Langel 2005). CPPs are able topenetrate biological membranes and as such trigger the movement ofvarious biomolecules across cell membranes into the cytoplasm and toimprove their intracellular routing, and hence facilitate interaction ofthe biolomolecule with the target. Examples of CPP include amongstothers: Tat, a nuclear transcriptional activator protein required forviral replication by HIV type1, penetratin, Kaposi fibroblast growthfactor (FGF) signal peptide sequence, integrin β3 signal peptidesequence; polyarginine peptide Args sequence, Guanine rich-moleculartransporters, sweet arrow peptide, etc.

Use of the System to make Genetically Modified Non-Transgenic Plants

In particular embodiments, the methods described herein are used tomodify endogenous genes or to modify their expression without thepermanent introduction into the genome of the plant of any foreign gene,including those encoding CRISPR components, so as to avoid the presenceof foreign DNA in the genome of the plant. This can be of interest asthe regulatory requirements for non-transgenic plants are less rigorous.

In particular embodiments, this is ensured by transient expression ofthe System components . In particular embodiments one or more of thecomponents are expressed on one or more viral vectors which producesufficient CRISPR-Cas protein, adenosine deaminase, and guide RNA toconsistently steadily ensure modification of a gene of interestaccording to a method described herein.

In particular embodiments, transient expression of System constructs isensured in plant protoplasts and thus not integrated into the genome.The limited window of expression can be sufficient to allow the Systemto ensure modification of a target gene as described herein.

In particular embodiments, the different components of the System areintroduced in the plant cell, protoplast or plant tissue eitherseparately or in mixture, with the aid of pariculate deliveringmolecules such as nanoparticles or CPP molecules as described hereinabove.

The expression of the System components can induce targeted modificationof the genome, by deaminase activity of the adenosine deaminase. Thedifferent strategies described herein above allow CRISPR-mediatedtargeted genome editing without requiring the introduction of theSystemt components into the plant genome. Components which aretransiently introduced into the plant cell are typically removed uponcrossing.

Plant Cultures and Regeneration

In particular embodiments, plant cells which have a modified genome andthat are produced or obtained by any of the methods described herein,can be cultured to regenerate a whole plant which possesses thetransformed or modified genotype and thus the desired phenotype.Conventional regeneration techniques are well known to those skilled inthe art. Particular examples of such regeneration techniques rely onmanipulation of certain phytohormones in a tissue culture growth medium,and typically relying on a biocide and/or herbicide marker which hasbeen introduced together with the desired nucleotide sequences. Infurther particular embodiments, plant regeneration is obtained fromcultured protoplasts, plant callus, explants, organs, pollens, embryosor parts thereof (see e.g. Evans et al. (1983), Handbook of Plant CellCulture, Klee et al (1987) Ann. Rev. of Plant Phys.).

In particular embodiments, transformed or improved plants as describedherein can be self-pollinated to provide seed for homozygous improvedplants of the invention (homozygous for the DNA modification) or crossedwith non-transgenic plants or different improved plants to provide seedfor heterozygous plants. Where a recombinant DNA was introduced into theplant cell, the resulting plant of such a crossing is a plant which isheterozygous for the recombinant DNA molecule. Both such homozygous andheterozygous plants obtained by crossing from the improved plants andcomprising the genetic modification (which can be a recombinant DNA) arereferred to herein as “progeny”. Progeny plants are plants descendedfrom the original transgenic plant and containing the genomemodification or recombinant DNA molecule introduced by the methodsprovided herein. Alternatively, genetically modified plants can beobtained by one of the methods described supra using the System wherebyno foreign DNA is incorporated into the genome. Progeny of such plants,obtained by further breeding may also contain the genetic modification.Breedings are performed by any breeding methods that are commonly usedfor different crops (e.g., Allard, Principles of Plant Breeding, JohnWiley & Sons, NY, U. of CA, Davis, Calif., 50-98 (1960).

Generation of Plants with Enhanced Agronomic Traits

The Systems provided herein can be used to introduce targeted A-G andT-C mutations. By co-expression of multiple targeting RNAs directed toachieve multiple modifications in a single cell, multiplexed genomemodification can be ensured. This technology can be used tohigh-precision engineering of plants with improved characteristics,including enhanced nutritional quality, increased resistance to diseasesand resistance to biotic and abiotic stress, and increased production ofcommercially valuable plant products or heterologous compounds.

In particular embodiments, the System as described herein is used tointroduce targeted A-G and T-C mutations. Such mutation can be anonsense mutation (e.g., premature stop codon) or a missense mutation(e.g., encoding different amino acid residue). This is of interest wherethe A-G and T-C mutations in certain endogenous genes can confer orcontribute to a desired trait.

The methods described herein generally result in the generation of“improved plants” in that they have one or more desirable traitscompared to the wildtype plant. In particular embodiments, the plants,plant cells or plant parts obtained are transgenic plants, comprising anexogenous DNA sequence incorporated into the genome of all or part ofthe cells of the plant. In particular embodiments, non-transgenicgenetically modified plants, plant parts or cells are obtained, in thatno exogenous DNA sequence is incorporated into the genome of any of theplant cells of the plant. In such embodiments, the improved plants arenon-transgenic. Where only the modification of an endogenous gene isensured and no foreign genes are introduced or maintained in the plantgenome, the resulting genetically modified crops contain no foreigngenes and can thus basically be considered non-transgenic.

In particular embodiments, the polynucleotides are delivered into thecell by a DNA virus (e.g., a geminivirus) or an RNA virus (e.g., atobravirus). In particular embodiments, the introducing steps includedelivering to the plant cell a T-DNA containing one or morepolynucleotide sequences encoding the CRISPR-Cas protein, the adenosinedeaminase, and the guide RNA, where the delivering is via Agrobacterium.The polynucleotide sequence encoding the components of the System can beoperably linked to a promoter, such as a constitutive promoter (e.g., acauliflower mosaic virus 35S promoter), or a cell specific or induciblepromoter. In particular embodiments, the polynucleotide is introduced bymicroprojectile bombardment. In particular embodiments, the methodfurther includes screening the plant cell after the introducing steps todetermine whether the expression of the gene of interest has beenmodified. In particular embodiments, the methods include the step ofregenerating a plant from the plant cell. In further embodiments, themethods include cross breeding the plant to obtain a genetically desiredplant lineage.

In particular embodiments of the methods described above, diseaseresistant crops are obtained by targeted mutation of diseasesusceptibility genes or genes encoding negative regulators (e.g. Mlogene) of plant defense genes. In a particular embodiment,herbicide-tolerant crops are generated by targeted substitution ofspecific nucleotides in plant genes such as those encoding acetolactatesynthase (ALS) and protoporphyrinogen oxidase (PPO). In particularembodiments drought and salt tolerant crops by targeted mutation ofgenes encoding negative regulators of abiotic stress tolerance, lowamylose grains by targeted mutation of Waxy gene, rice or other grainswith reduced rancidity by targeted mutation of major lipase genes inaleurone layer, etc. In particular embodiments. A more extensive list ofendogenous genes encoding a traits of interest are listed below.

Use of System to Modify Polyploid Plants

Many plants are polyploid, which means they carry duplicate copies oftheir genomes—sometimes as many as six, as in wheat. The methodsaccording to the present invention, which make use of the System can be“multiplexed” to affect all copies of a gene, or to target dozens ofgenes at once. For instance, in particular embodiments, the methods ofthe present invention are used to simultaneously ensure a loss offunction mutation in different genes responsible for suppressingdefences against a disease. In particular embodiments, the methods ofthe present invention are used to simultaneously suppress the expressionof the TaMLO-A1, TaMLO-B1 and TaMLO-D1 nucleic acid sequence in a wheatplant cell and regenerating a wheat plant therefrom, in order to ensurethat the wheat plant is resistant to powdery mildew (see alsoWO2015109752).

Examplary Genes Conferring Agronomic Traits

In particular embodiments, the invention encompasses methods whichinvolve targeted A-G and T-C mutations in endogenous genes and theirregulatory elements, such as listed below:

1. Genes that Confer Resistance to Pests or Diseases:

Plant disease resistance genes. A plant can be transformed with clonedresistance genes to engineer plants that are resistant to specificpathogen strains. See, e.g., Jones et al., Science 266:789 (1994)(cloning of the tomato Cf-9 gene for resistance to Cladosporium fulvum);Martin et al., Science 262:1432 (1993) (tomato Pto gene for resistanceto Pseudomonas syringae pv. tomato encodes a protein kinase); Mindrinoset al., Cell 78:1089 (1994) (Arabidops may be RSP2 gene for resistanceto Pseudomonas syringae). A plant gene that is upregulated or downregulated during pathogen infection can be engineered for pathogenresistance. See, e.g., Thomazella et al., bioRxiv 064824; doi:doi.org/10.1101/064824 Epub. Jul. 23, 2016 (tomato plants with deletionsin the S1DMR6-1 which is normally upregulated during pathogeninfection).

Genes conferring resistance to a pest, such as soybean cyst nematode.See e.g., PCT Application WO 96/30517; PCT Application WO 93/19181.

Bacillus thuringiensis proteins see, e.g., Geiser et al., Gene 48:109(1986).

Lectins, see, for example, Van Damme et al., Plant Molec. Biol. 24:25(1994.

Vitamin-binding protein, such as avidin, see PCT application US93/06487,teaching the use of avidin and avidin homologues as larvicides againstinsect pests.

Enzyme inhibitors such as protease or proteinase inhibitors or amylaseinhibitors. See, e.g., Abe et al., J. Biol. Chem. 262:16793 (1987), Huubet al., Plant Molec. Biol. 21:985 (1993)), Sumitani et al., Biosci.Biotech. Biochem. 57:1243 (1993) and U.S. Pat. No. 5,494,813.

Insect-specific hormones or pheromones such as ecdysteroid or juvenilehormone, a variant thereof, a mimetic based thereon, or an antagonist oragonist thereof. See, for example Hammock et al., Nature 344:458 (1990).

Insect-specific peptides or neuropeptides which, upon expression,disrupts the physiology of the affected pest. For example Regan, J.Biol. Chem. 269:9 (1994) and Pratt et al., Biochem. Biophys. Res. Comm.163:1243 (1989). See also U.S. Pat. No. 5,266,317.

Insect-specific venom produced in nature by a snake, a wasp, or anyother organism. For example, see Pang et al., Gene 116: 165 (1992).

Enzymes responsible for a hyperaccumulation of a monoterpene, asesquiterpene, a steroid, hydroxamic acid, a phenylpropanoid derivativeor another nonprotein molecule with insecticidal activity.

Enzymes involved in the modification, including the post-translationalmodification, of a biologically active molecule; for example, aglycolytic enzyme, a proteolytic enzyme, a lipolytic enzyme, a nuclease,a cyclase, a transaminase, an esterase, a hydrolase, a phosphatase, akinase, a phosphorylase, a polymerase, an elastase, a chitinase and aglucanase, whether natural or synthetic. See PCT application WO93/02197,Kramer et al., Insect Biochem. Molec. Biol. 23:691 (1993) and Kawallecket al., Plant Molec. Biol. 21:673 (1993).

Molecules that stimulates signal transduction. For example, see Botellaet al., Plant Molec. Biol. 24:757 (1994), and Griess et al., PlantPhysiol. 104:1467 (1994).

Viral-invasive proteins or a complex toxin derived therefrom. See Beachyet al., Ann. rev. Phytopathol. 28:451 (1990).

Developmental-arrestive proteins produced in nature by a pathogen or aparasite. See Lamb et al., Bio/Technology 10:1436 (1992) and Toubart etal., Plant J. 2:367 (1992).

A developmental-arrestive protein produced in nature by a plant. Forexample, Logemann et al., Bio/Technology 10:305 (1992).

In plants, pathogens are often host-specific. For example, some Fusariumspecies will causes tomato wilt but attacks only tomato, and otherFusarium species attack only wheat. Plants have existing and induceddefenses to resist most pathogens. Mutations and recombination eventsacross plant generations lead to genetic variability that gives rise tosusceptibility, especially as pathogens reproduce with more frequencythan plants. In plants there can be non-host resistance, e.g., the hostand pathogen are incompatible or there can be partial resistance againstall races of a pathogen, typically controlled by many genes and/or alsocomplete resistance to some races of a pathogen but not to other races.Such resistance is typically controlled by a few genes. Using methodsand components of the System, a new tool now exists to induce specificmutations in anticipation hereon. Accordingly, one can analyze thegenome of sources of resistance genes, and in plants having desiredcharacteristics or traits, use the method and components of the Systemto induce the rise of resistance genes. The present systems can do sowith more precision than previous mutagenic agents and hence accelerateand improve plant breeding programs.

2. Genes involved in Plant Diseases, such as those listed in WO2013046247:

Rice diseases: Magnaporthe grisea, Cochliobolus miyabeanus, Rhizoctoniasolani, Gibberella fujikuroi; Wheat diseases: Erysiphe graminis,Fusarium graminearum, F. avenaceum, F. culmorum, Microdochium nivale,Puccinia striiformis, P. graminis, P. recondita, Micronectriella nivale,Typhula sp., Ustilago tritici, Tilletia caries, Pseudocercosporellaherpotrichoides, Mycosphaerella graminicola, Stagonospora nodorum,Pyrenophora tritici-repentis; Barley diseases: Erysiphe graminis,Fusarium graminearum, F. avenaceum, F. culmorum, Microdochium nivale,Puccinia striiformis, P. graminis, P. hordei, Ustilago nuda,Rhynchosporium secalis, Pyrenophora teres, Cochliobolus sativus,Pyrenophora graminea, Rhizoctonia solani; Maize diseases: Ustilagomaydis, Cochliobolus heterostrophus, Gloeocercospora sorghi, Pucciniapolysora, Cercospora zeae-maydis, Rhizoctonia solani;

Citrus diseases: Diaporthe citri, Elsinoe fawcetti, Penicilliumdigitatum, P. italicum, Phytophthora parasitica, Phytophthoracitrophthora; Apple diseases: Monilinia mali, Valsa ceratosperma,Podosphaera leucotricha, Alternaria alternata apple pathotype, Venturiainaequalis, Colletotrichum acutatum, Phytophtora cactorum;

Pear diseases: Venturia nashicola, V. pirina, Alternaria alternataJapanese pear pathotype, Gymnosporangium haraeanum, Phytophtoracactorum;

Peach diseases: Monilinia fructicola, Cladosporium carpophilum,Phomopsis sp.;

Grape diseases: Elsinoe ampelina, Glomerella cingulata, Uninula necator,Phakopsora ampelopsidis, Guignardia bidwellii, Plasmopara viticola;

Persimmon diseases: Gloesporium kaki, Cercospora kaki, Mycosphaerelanawae;

Gourd diseases: Colletotrichum lagenarium, Sphaerotheca fuliginea,Mycosphaerella melonis, Fusarium oxysporum, Pseudoperonospora cubensis,Phytophthora sp., Pythium sp.;

Tomato diseases: Alternaria solani, Cladosporium fulvum, Phytophthorainfestans; Pseudomonas syringae pv. Tomato; Phytophthora capsici;Xanthomonas

Eggplant diseases: Phomopsis vexans, Erysiphe cichoracearum;Brassicaceous vegetable diseases: Alternaria japonica, Cercosporellabrassicae, Plasmodiophora brassicae, Peronospora parasitica;

Welsh onion diseases: Puccinia allii, Peronospora destructor;

Soybean diseases: Cercospora kikuchii, Elsinoe glycines, Diaporthephaseolorum var. sojae, Septoria glycines, Cercospora sojina, Phakopsorapachyrhizi, Phytophthora sojae, Rhizoctonia solani, Corynesporacasiicola, Sclerotinia sclerotiorum;

Kidney bean diseases: Colletrichum lindemthianum;

Peanut diseases: Cercospora personata, Cercospora arachidicola,Sclerotium rolfsii;

Pea diseases pea: Erysiphe pisi;

Potato diseases: Alternaria solani, Phytophthora infestans, Phytophthoraerythroseptica, Spongospora subterranean, f. sp. Subterranean;

Strawberry diseases: Sphaerotheca humuli, Glomerella cingulata;

Tea diseases: Exobasidium reticulatum, Elsinoe leucospila,Pestalotiopsis sp., Colletotrichum theae-sinensis;

Tobacco diseases: Alternaria longipes, Erysiphe cichoracearum,Colletotrichum tabacum, Peronospora tabacina, Phytophthora nicotianae;

Rapeseed diseases: Sclerotinia sclerotiorum, Rhizoctonia solani;

Cotton diseases: Rhizoctonia solani;

Beet diseases: Cercospora beticola, Thanatephorus cucumeris,Thanatephorus cucumeris, Aphanomyces cochlioides;

Rose diseases: Diplocarpon rosae, Sphaerotheca pannosa, Peronosporasparsa;

Diseases of chrysanthemum and asteraceae: Bremia lactuca, Septoriachrysanthemi-indici, Puccinia horiana;

Diseases of various plants: Pythium aphanidermatum, Pythium debarianum,Pythium graminicola, Pythium irregulare, Pythium ultimum, Botrytiscinerea, Sclerotinia sclerotiorum;

Radish diseases: Alternaria brassicicola;

Zoysia diseases: Sclerotinia homeocarpa, Rhizoctonia solani;

Banana diseases: Mycosphaerella fijiensis, Mycosphaerella musicola;

Sunflower diseases: Plasmopara halstedii;

Seed diseases or diseases in the initial stage of growth of variousplants caused by Aspergillus spp., Penicillium spp., Fusarium spp.,Gibberella spp., Tricoderma spp., Thielaviopsis spp., Rhizopus spp.,Mucor spp., Corticium spp., Rhoma spp., Rhizoctonia spp., Diplodia spp.,or the like;

Virus diseases of various plants mediated by Polymixa spp., Olpidiumspp., or the like.

3. Examples of Genes that Confer Resistance to Herbicides:

Resistance to herbicides that inhibit the growing point or meristem,such as an imidazolinone or a sulfonylurea, for example, by Lee et al.,EMBO J. 7:1241 (1988), and Miki et al., Theor. Appl. Genet. 80:449(1990), respectively.

Glyphosate tolerance (resistance conferred by, e.g., mutant5-enolpyruvylshikimate-3-phosphate synthase (EPSPs) genes, aroA genesand glyphosate acetyl transferase (GAT) genes, respectively), orresistance to other phosphono compounds such as by glufosinate(phosphinothricin acetyl transferase (PAT) genes from Streptomycesspecies, including Streptomyces hygroscopicus and Streptomycesviridichromogenes), and to pyridinoxy or phenoxy proprionic acids andcyclohexones by ACCase inhibitor-encoding genes. See, for example, U.S.Pat. Nos. 4,940,835 and 6,248,876, 4,769,061, EP No. 0 333 033 and U.S.Pat No. 4,975,374. See also EP No. 0242246, DeGreef et al.,Bio/Technology 7:61 (1989), Marshall et al., Theor. Appl. Genet. 83:435(1992), WO 2005012515 to Castle et. al. and WO 2005107437.

Resistance to herbicides that inhibit photosynthesis, such as a triazine(psbA and gs+ genes) or a benzonitrile (nitrilase gene), and glutathioneS-transferase in Przibila et al., Plant Cell 3:169 (1991), U.S. Pat. No.4,810,648, and Hayes et al., Biochem. J. 285: 173 (1992).

Genes encoding Enzymes detoxifying the herbicide or a mutant glutaminesynthase enzyme that is resistant to inhibition, e.g. n U.S. patentapplication Ser. No. 11/760,602. Or a detoxifying enzyme is an enzymeencoding a phosphinothricin acetyltransferase (such as the bar or patprotein from Streptomyces species). Phosphinothricin acetyltransferasesare for example described in U.S. Pat. Nos. 5,561,236; 5,648,477;5,646,024; 5,273,894; 5,637,489; 5,276,268; 5,739,082; 5,908,810 and7,112,665.

Hydroxyphenylpyruvatedioxygenases (HPPD) inhibitors, naturally occurringHPPD resistant enzymes, or genes encoding a mutated or chimeric HPPDenzyme as described in WO 96/38567, WO 99/24585, and WO 99/24586, WO2009/144079, WO 2002/046387, or U.S. Pat. No. 6,768,044.

4. Examples of Genes involved in Abiotic Stress Tolerance:

Transgene capable of reducing the expression and/or the activity ofpoly(ADP-ribose) polymerase (PARP) gene in the plant cells or plants asdescribed in WO 00/04173 or, WO/2006/045633.

Transgenes capable of reducing the expression and/or the activity of thePARG encoding genes of the plants or plants cells, as described e.g. inWO 2004/090140.

Transgenes coding for a plant-functional enzyme of the nicotineamideadenine dinucleotide salvage synthesis pathway including nicotinamidase,nicotinate phosphoribosyltransferase, nicotinic acid mononucleotideadenyl transferase, nicotinamide adenine dinucleotide synthetase ornicotine amide phosphorybosyltransferase as described e.g. in EP04077624.7, WO 2006/133827, PCT/EP07/002,433, EP 1999263, or WO2007/107326.

Enzymes involved in carbohydrate biosynthesis include those described ine.g. EP 0571427, WO 95/04826, EP 0719338, WO 96/15248, WO 96/19581, WO96/27674, WO 97/11188, WO 97/26362, WO 97/32985, WO 97/42328, WO97/44472, WO 97/45545, WO 98/27212, WO 98/40503, WO99/58688, WO99/58690, WO 99/58654, WO 00/08184, WO 00/08185, WO 00/08175, WO00/28052, WO 00/77229, WO 01/12782, WO 01/12826, WO 02/101059, WO03/071860, WO 2004/056999, WO 2005/030942, WO 2005/030941, WO2005/095632, WO 2005/095617, WO 2005/095619, WO 2005/095618, WO2005/123927, WO 2006/018319, WO 2006/103107, WO 2006/108702, WO2007/009823, WO 00/22140, WO 2006/063862, WO 2006/072603, WO 02/034923,EP 06090134.5, EP 06090228.5, EP 06090227.7, EP 07090007.1, EP07090009.7, WO 01/14569, WO 02/79410, WO 03/33540, WO 2004/078983, WO01/19975, WO 95/26407, WO 96/34968, WO 98/20145, WO 99/12950, WO99/66050, WO 99/53072, U.S. Pat. No. 6,734,341, WO 00/11192, WO98/22604, WO 98/32326, WO 01/98509, WO 01/98509, WO 2005/002359, U.S.Pat. Nos. 5,824,790, 6,013,861, WO 94/04693, WO 94/09144, WO 94/11520,WO 95/35026 or WO 97/20936 or enzymes involved in the production ofpolyfructose, especially of the inulin and levan-type, as disclosed inEP 0663956, WO 96/01904, WO 96/21023, WO 98/39460, and WO 99/24593, theproduction of alpha-1,4-glucans as disclosed in WO 95/31553, US2002031826, U.S. Pat. Nos. 6,284,479, 5,712,107, WO 97/47806, WO97/47807, WO 97/47808 and WO 00/14249, the production of alpha-1,6branched alpha-1,4-glucans, as disclosed in WO 00/73422, the productionof alternan, as disclosed in e.g. WO 00/47727, WO 00/73422, EP06077301.7, U.S. Pat. No. 5,908,975 and EP 0728213, the production ofhyaluronan, as for example disclosed in WO 2006/032538, WO 2007/039314,WO 2007/039315, WO 2007/039316, JP 2006304779, and WO 2005/012529.

Genes that improve drought resistance. For example, WO 2013122472discloses that the absence or reduced level of functional UbiquitinProtein Ligase protein (UPL) protein, more specifically, UPL3, leads toa decreased need for water or improved resistance to drought of saidplant. Other examples of transgenic plants with increased droughttolerance are disclosed in, for example, US 2009/0144850, US2007/0266453, and WO 2002/083911. US2009/0144850 describes a plantdisplaying a drought tolerance phenotype due to altered expression of aDR02 nucleic acid. US 2007/0266453 describes a plant displaying adrought tolerance phenotype due to altered expression of a DR03 nucleicacid and WO 2002/08391 1 describes a plant having an increased toleranceto drought stress due to a reduced activity of an ABC transporter whichis expressed in guard cells. Another example is the work by Kasuga andco-authors (1999), who describe that overexpression of cDNA encodingDREB1 A in transgenic plants activated the expression of many stresstolerance genes under normal growing conditions and resulted in improvedtolerance to drought, salt loading, and freezing. However, theexpression of DREB1A also resulted in severe growth retardation undernormal growing conditions (Kasuga (1999) Nat Biotechnol 17(3) 287-291).

In further particular embodiments, crop plants can be improved byinfluencing specific plant traits. For example, by developingpesticide-resistant plants, improving disease resistance in plants,improving plant insect and nematode resistance, improving plantresistance against parasitic weeds, improving plant drought tolerance,improving plant nutritional value, improving plant stress tolerance,avoiding self-pollination, plant forage digestibility biomass, grainyield etc. A few specific non-limiting examples are providedhereinbelow.

In addition to targeted mutation of single genes, System can be designedto allow targeted mutation of multiple genes, deletion of chromosomalfragment, site-specific integration of transgene, site-directedmutagenesis in vivo, and precise gene replacement or allele swapping inplants. Therefore, the methods described herein have broad applicationsin gene discovery and validation, mutational and cisgenic breeding, andhybrid breeding. These applications facilitate the production of a newgeneration of genetically modified crops with various improved agronomictraits such as herbicide resistance, disease resistance, abiotic stresstolerance, high yield, and superior quality.

Use of System to Create Male Sterile Plants

Hybrid plants typically have advantageous agronomic traits compared toinbred plants. However, for self-pollinating plants, the generation ofhybrids can be challenging. In different plant types, genes have beenidentified which are important for plant fertility, more particularlymale fertility. For instance, in maize, at least two genes have beenidentified which are important in fertility (Amitabh MohantyInternational Conference on New Plant Breeding Molecular TechnologiesTechnology Development And Regulation, Oct. 9-10, 2014, Jaipur, India;Svitashev et al. Plant Physiol. 2015 October; 169(2):931-45; Djukanovicet al. Plant J. 2013 December; 76(5):888-99). The methods and systemsprovided herein can be used to target genes required for male fertilityso as to generate male sterile plants which can easily be crossed togenerate hybrids. In particular embodiments, the System provided hereinis used for targeted mutagenesis of the cytochrome P450-like gene (MS26)or the meganuclease gene (MS45) thereby conferring male sterility to themaize plant. Maize plants which are as such genetically altered can beused in hybrid breeding programs.

Increasing the Fertility Stage in Plants

In particular embodiments, the methods and systems provided herein areused to prolong the fertility stage of a plant such as of a rice plant.For instance, a rice fertility stage gene such as Ehd3 can be targetedin order to generate a mutation in the gene and plantlets can beselected for a prolonged regeneration plant fertility stage (asdescribed in CN 104004782)

Use of System to Generate Genetic Variation in a Crop of Interest

The availability of wild germplasm and genetic variations in crop plantsis the key to crop improvement programs, but the available diversity ingermplasms from crop plants is limited. The present invention envisagesmethods for generating a diversity of genetic variations in a germplasmof interest. In this application of the System a library of guide RNAstargeting different locations in the plant genome is provided and isintroduced into plant cells together with the CRISPR-Cas protein andadenosine deaminase. In this way a collection of genome-scale pointmutations and gene knock-outs can be generated. In particularembodiments, the methods comprise generating a plant part or plant fromthe cells so obtained and screening the cells for a trait of interest.The target genes can include both coding and non-coding regions. Inparticular embodiments, the trait is stress tolerance and the method isa method for the generation of stress-tolerant crop varieties

Use of AD-Functionalized CRISPR to Affect Fruit-Ripening

Ripening is a normal phase in the maturation process of fruits andvegetables. Only a few days after it starts it renders a fruit orvegetable inedible. This process brings significant losses to bothfarmers and consumers. In particular embodiments, the methods of thepresent invention are used to reduce ethylene production. This isensured by ensuring one or more of the following: a. Suppression of ACCsynthase gene expression. ACC (1-aminocyclopropane-1-carboxylic acid)synthase is the enzyme responsible for the conversion ofS-adenosylmethionine (SAM) to ACC; the second to the last step inethylene biosynthesis. Enzyme expression is hindered when an antisense(“mirror-image”) or truncated copy of the synthase gene is inserted intothe plant's genome; b. Insertion of the ACC deaminase gene. The genecoding for the enzyme is obtained from Pseudomonas chlororaphis, acommon nonpathogenic soil bacterium. It converts ACC to a differentcompound thereby reducing the amount of ACC available for ethyleneproduction; c. Insertion of the SAM hydrolase gene. This approach issimilar to ACC deaminase wherein ethylene production is hindered whenthe amount of its precursor metabolite is reduced; in this case SAM isconverted to homoserine. The gene coding for the enzyme is obtained fromE. coli T3 bacteriophage and d. Suppression of ACC oxidase geneexpression. ACC oxidase is the enzyme which catalyzes the oxidation ofACC to ethylene, the last step in the ethylene biosynthetic pathway.Using the methods described herein, down regulation of the ACC oxidasegene results in the suppression of ethylene production, thereby delayingfruit ripening. In particular embodiments, additionally or alternativelyto the modifications described above, the methods described herein areused to modify ethylene receptors, so as to interfere with ethylenesignals obtained by the fruit. In particular embodiments, expression ofthe ETR1 gene, encoding an ethylene binding protein is modified, moreparticularly suppressed. In particular embodiments, additionally oralternatively to the modifications described above, the methodsdescribed herein are used to modify expression of the gene encodingPolygalacturonase (PG), which is the enzyme responsible for thebreakdown of pectin, the substance that maintains the integrity of plantcell walls. Pectin breakdown occurs at the start of the ripening processresulting in the softening of the fruit. Accordingly, in particularembodiments, the methods described herein are used to introduce amutation in the PG gene or to suppress activation of the PG gene inorder to reduce the amount of PG enzyme produced thereby delaying pectindegradation.

Thus in particular embodiments, the methods comprise the use of theSystem to ensure one or more modifications of the genome of a plant cellsuch as described above, and regenerating a plant therefrom. Inparticular embodiments, the plant is a tomato plant.

Increasing Storage Life of Plants

In particular embodiments, the methods of the present invention are usedto modify genes involved in the production of compounds which affectstorage life of the plant or plant part. More particularly, themodification is in a gene that prevents the accumulation of reducingsugars in potato tubers. Upon high-temperature processing, thesereducing sugars react with free amino acids, resulting in brown,bitter-tasting products and elevated levels of acrylamide, which is apotential carcinogen. In particular embodiments, the methods providedherein are used to reduce or inhibit expression of the vacuolarinvertase gene (VInv), which encodes a protein that breaks down sucroseto glucose and fructose (Clasen et al. DOI: 10.1111/pbi.12370).

The Use of the System to Ensure a Value Added Trait

In particular embodiments the System is used to produce nutritionallyimproved agricultural crops. In particular embodiments, the methodsprovided herein are adapted to generate “functional foods”, i.e. amodified food or food ingredient that may provide a health benefitbeyond the traditional nutrients it contains and or “nutraceutical”,i.e. substances that may be considered a food or part of a food andprovides health benefits, including the prevention and treatment ofdisease. In particular embodiments, the nutraceutical is useful in theprevention and/or treatment of one or more of cancer, diabetes,cardiovascular disease, and hypertension.

Examples of nutritionally improved crops include (Newell-McGloughlin,Plant Physiology, July 2008, Vol. 147, pp. 939-953):

Modified protein quality, content and/or amino acid composition, such ashave been described for Bahiagrass (Luciani et al. 2005, FloridaGenetics Conference Poster), Canola (Roesler et al., 1997, Plant Physiol113 75-81), Maize (Cromwell et al, 1967, 1969 J Anim Sci 26 1325-1331,O′Quin et al. 2000 J Anim Sci 78 2144-2149, Yang et al. 2002, TransgenicRes 11 11-20, Young et al. 2004, Plant J 38 910-922), Potato (Yu J andAo, 1997 Acta Bot Sin 39 329-334; Chakraborty et al. 2000, Proc NatlAcad Sci USA 97 3724-3729; Li et al. 2001) Chin Sci Bull 46 482-484,Rice (Katsube et al. 1999, Plant Physiol 120 1063-1074), Soybean(Dinkins et al. 2001, Rapp 2002, In Vitro Cell Dev Biol Plant 37742-747), Sweet Potato (Egnin and Prakash 1997, In Vitro Cell Dev Biol33 52A).

Essential amino acid content, such as has been described for Canola(Falco et al. 1995, Bio/Technology 13 577-582), Lupin (White et al.2001, J Sci Food Agric 81 147-154), Maize (Lai and Messing, 2002, Agbios2008 GM crop database (Mar. 11, 2008)), Potato (Zeh et al. 2001, PlantPhysiol 127 792-802), Sorghum (Zhao et al. 2003, Kluwer AcademicPublishers, Dordrecht, The Netherlands, pp 413-416), Soybean (Falco etal. 1995 Bio/Technology 13 577-582; Galili et al. 2002 Crit Rev PlantSci 21 167-204).

Oils and Fatty acids such as for Canola (Dehesh et al. (1996) Plant J 9167-172 [PubMed]; Del Vecchio (1996) INFORM International News on Fats,Oils and Related Materials 7 230-243; Roesler et al. (1997) PlantPhysiol 113 75-81 [PMC free article] [PubMed]; Froman and Ursin (2002,2003) Abstracts of Papers of the American Chemical Society 223 U35;James et al. (2003) Am J Clin Nutr 77 1140-1145 [PubMed]; Agbios (2008,above); coton (Chapman et al. (2001). J Am Oil Chem Soc 78 941-947; Liuet al. (2002) J Am Coll Nutr 21 205S-211S [PubMed]; O′Neill (2007)Australian Life Scientist.www.biotechnews.com.au/index.php/id;866694817;fp;4;fpid;2 (Jun. 17,2008), Linseed (Abbadi et al., 2004, Plant Cell 16: 2734-2748), Maize(Young et al., 2004, Plant J 38 910-922), oil palm (Jalani et al. 1997,J Am Oil Chem Soc 74 1451-1455; Parveez, 2003, AgBiotechNet 113 1-8),Rice (Anai et al., 2003, Plant Cell Rep 21 988-992), Soybean (Reddy andThomas, 1996, Nat Biotechnol 14 639-642; Kinney and Kwolton, 1998,Blackie Academic and Professional, London, pp 193-213), Sunflower(Arcadia, Biosciences 2008)

Carbohydrates, such as Fructans described for Chicory (Smeekens (1997)Trends Plant Sci 2 286-287, Sprenger et al. (1997) FEBS Lett 400355-358, Sévenier et al. (1998) Nat Biotechnol 16 843-846), Maize (Caimiet al. (1996) Plant Physiol 110 355-363), Potato (Hellwege et al., 1997Plant J 12 1057-1065), Sugar Beet (Smeekens et al. 1997, above), Inulin,such as described for Potato (Hellewege et al. 2000, Proc Natl Acad SciUSA 97 8699-8704), Starch, such as described for Rice (Schwall et al.(2000) Nat Biotechnol 18 551-554, Chiang et al. (2005) Mol Breed 15125-143),

Vitamins and carotenoids, such as described for Canola (Shintani andDellaPenna (1998) Science 282 2098-2100), Maize (Rocheford et al. (2002). J Am Coll Nutr 21 191S-198S, Cahoon et al. (2003) Nat Biotechnol 211082-1087, Chen et al. (2003) Proc Natl Acad Sci USA 100 3525-3530),Mustardseed (Shewmaker et al. (1999) Plant J 20 401-412, Potato (Ducreuxet al., 2005, J Exp Bot 56 81-89), Rice (Ye et al. (2000) Science 287303-305, Strawberry (Agius et al. (2003), Nat Biotechnol 21 177-181),Tomato (Rosati et al. (2000) Plant J 24 413-419, Fraser et al. (2001) JSci Food Agric 81 822-827, Mehta et al. (2002) Nat Biotechnol 20613-618, Diaz de la Garza et al. (2004) Proc Natl Acad Sci USA 10113720-13725, Enfissi et al. (2005) Plant Biotechnol J 3 17-27,DellaPenna (2007) Proc Natl Acad Sci USA 104 3675-3676.

Functional secondary metabolites, such as described for Apple(stilbenes, Szankowski et al. (2003) Plant Cell Rep 22: 141-149),Alfalfa (resveratrol, Hipskind and Paiva (2000) Mol Plant MicrobeInteract 13 551-562), Kiwi (resveratrol, Kobayashi et al. (2000) PlantCell Rep 19 904-910), Maize and Soybean (flavonoids, Yu et al. (2000)Plant Physiol 124 781-794), Potato (anthocyanin and alkaloid glycoside,Lukaszewicz et al. (2004) J Agric Food Chem 52 1526-1533), Rice(flavonoids & resveratrol, Stark-Lorenzen et al. (1997) Plant Cell Rep16 668-673, Shin et al. (2006) Plant Biotechnol J 4 303-315), Tomato(+resveratrol, chlorogenic acid, flavonoids, stilbene; Rosati et al.(2000) above, Muir et al. (2001) Nature 19 470-474, Niggeweg et al.(2004) Nat Biotechnol 22 746-754, Giovinazzo et al. (2005) PlantBiotechnol J 3 57-69), wheat (caffeic and ferulic acids, resveratrol;United Press International (2002)); and

Mineral availabilities such as described for Alfalfa (phytase,Austin-Phillips et al. (1999) www.molecularfarming.com/nonmedical.html),Lettuse (iron, Goto et al. (2000) Theor Appl Genet 100 658-664), Rice(iron, Lucca et al. (2002) J Am Coll Nutr 21 184S-190S), Maize, Soybeanand wheate (phytase, Drakakaki et al. (2005) Plant Mol Biol 59 869-880,Denbow et al. (1998) Poult Sci 77 878-881, Brinch-Pedersen et al. (2000)Mol Breed 6 195-206).

In particular embodiments, the value-added trait is related to theenvisaged health benefits of the compounds present in the plant. Forinstance, in particular embodiments, the value-added crop is obtained byapplying the methods of the invention to ensure the modification of orinduce/increase the synthesis of one or more of the following compounds:

-   -   Carotenoids, such as α-Carotene present in carrots which        Neutralizes free radicals that may cause damage to cells or        β-Carotene present in various fruits and vegetables which        neutralizes free radicals;    -   Lutein present in green vegetables which contributes to        maintenance of healthy vision;    -   Lycopene present in tomato and tomato products, which is        believed to reduce the risk of prostate cancer;    -   Zeaxanthin, present in citrus and maize, which contributes to        maintenance of healthy vision    -   Dietary fiber such as insoluble fiber present in wheat bran        which may reduce the risk of breast and/or colon cancer and        β-Glucan present in oat, soluble fiber present in Psylium and        whole cereal grains which may reduce the risk of cardiovascular        disease (CVD);    -   Fatty acids, such as ω-3 fatty acids which may reduce the risk        of CVD and improve mental and visual functions, Conjugated        linoleic acid, which may improve body composition, may decrease        risk of certain cancers and GLA which may reduce inflammation        risk of cancer and CVD, may improve body composition;    -   Flavonoids such as Hydroxycinnamates, present in wheat which        have Antioxidant-like activities, may reduce risk of        degenerative diseases, flavonols, catechins and tannins present        in fruits and vegetables which neutralize free radicals and may        reduce risk of cancer;    -   Glucosinolates, indoles, isothiocyanates, such as Sulforaphane,        present in Cruciferous vegetables (broccoli, kale), horseradish,        which neutralize free radicals, may reduce risk of cancer;    -   Phenolics, such as stilbenes present in grape which May reduce        risk of degenerative diseases, heart disease, and cancer, may        have longevity effect and caffeic acid and ferulic acid present        in vegetables and citrus which have Antioxidant-like activities,        may reduce risk of degenerative diseases, heart disease, and eye        disease, and epicatechin present in cacao which has        Antioxidant-like activities, may reduce risk of degenerative        diseases and heart disease;    -   Plant stanols/sterols present in maize, soy, wheat and wooden        oils which May reduce risk of coronary heart disease by lowering        blood cholesterol levels;    -   Fructans, inulins, fructo-oligosaccharides present in Jerusalem        artichoke, shallot, onion powder which may improve        gastrointestinal health;    -   Saponins present in soybean, which may lower LDL cholesterol;    -   Soybean protein present in soybean which may reduce risk of        heart disease;    -   Phytoestrogens such as isoflavones present in soybean which May        reduce menopause symptoms, such as hot flashes, may reduce        osteoporosis and CVD and lignans present in flax, rye and        vegetables, which May protect against heart disease and some        cancers, may lower LDL cholesterol, total cholesterol;    -   Sulfides and thiols such as diallyl sulphide present in onion,        garlic, olive, leek and scallon and Allyl methyl trisulfide,        dithiolthiones present in cruciferous vegetables which may lower        LDL cholesterol, helps to maintain healthy immune system; and    -   Tannins, such as proanthocyanidins, present in cranberry, cocoa,        which may improve urinary tract health, may reduce risk of CVD        and high blood pressure.

In addition, the methods of the present invention also envisagemodifying protein/starch functionality, shelf life, taste/aesthetics,fiber quality, and allergen, antinutrient, and toxin reduction traits.

Accordingly, the invention encompasses methods for producing plants withnutritional added value, said methods comprising introducing into aplant cell a gene encoding an enzyme involved in the production of acomponent of added nutritional value using the System as describedherein and regenerating a plant from said plant cell, said plantcharacterized in an increase expression of said component of addednutritional value. In particular embodiments, the System is used tomodify the endogenous synthesis of these compounds indirectly, e.g. bymodifying one or more transcription factors that controls the metabolismof this compound. Methods for introducing a gene of interest into aplant cell and/or modifying an endogenous gene using the System aredescribed herein above.

Some specific examples of modifications in plants that have beenmodified to confer value-added traits are: plants with modified fattyacid metabolism, for example, by transforming a plant with an antisensegene of stearyl-ACP desaturase to increase stearic acid content of theplant. See Knultzon et al., Proc. Natl. Acad. Sci. U.S.A. 89:2624(1992). Another example involves decreasing phytate content, for exampleby cloning and then reintroducing DNA associated with the single allelewhich may be responsible for maize mutants characterized by low levelsof phytic acid. See Raboy et al, Maydica 35:383 (1990).

Similarly, expression of the maize (Zea mays) Tfs C1 and R, whichregulate the production of flavonoids in maize aleurone layers under thecontrol of a strong promoter, resulted in a high accumulation rate ofanthocyanins in Arabidopsis (Arabidopsis thaliana), presumably byactivating the entire pathway (Bruce et al., 2000, Plant Cell 12:65-80).DellaPenna (Welsch et al., 2007 Annu Rev Plant Biol 57: 711-73 8) foundthat Tf RAP2.2 and its interacting partner SINAT2 increasedcarotenogenesis in Arabidopsis leaves. Expressing the Tf Dof1 inducedthe up-regulation of genes encoding enzymes for carbon skeletonproduction, a marked increase of amino acid content, and a reduction ofthe Glc level in transgenic Arabidopsis (Yanagisawa, 2004 Plant CellPhysiol 45: 386-391), and the DOF Tf AtDof1.1 (OBP2) up-regulated allsteps in the glucosinolate biosynthetic pathway in Arabidopsis (Skiryczet al., 2006 Plant J 47: 10-24).

Reducing Allergen in Plants

In particular embodiments the methods provided herein are used togenerate plants with a reduced level of allergens, making them safer forthe consumer. In particular embodiments, the methods comprise modifyingexpression of one or more genes responsible for the production of plantallergens. For instance, in particular embodiments, the methods comprisedown-regulating expression of a Lol p5 gene in a plant cell, such as aryegrass plant cell and regenerating a plant therefrom so as to reduceallergenicity of the pollen of said plant (Bhalla et al. 1999, Proc.Natl. Acad. Sci. USA Vol. 96: 11676-11680).

Peanut allergies and allergies to legumes generally are a real andserious health concern. The System of the present invention can be usedto identify and then mutate genes encoding allergenic proteins of suchlegumes. Without limitation as to such genes and proteins, Nicolaou etal. identifies allergenic proteins in peanuts, soybeans, lentils, peas,lupin, green beans, and mung beans. See, Nicolaou et al., CurrentOpinion in Allergy and Clinical Immunology 2011; 11(3):222).

Screening Methods for Endogenous Genes of Interest

The methods provided herein further allow the identification of genes ofvalue encoding enzymes involved in the production of a component ofadded nutritional value or generally genes affecting agronomic traits ofinterest, across species, phyla, and plant kingdom. By selectivelytargeting e.g. genes encoding enzymes of metabolic pathways in plantsusing the System as described herein, the genes responsible for certainnutritional aspects of a plant can be identified. Similarly, byselectively targeting genes which may affect a desirable agronomictrait, the relevant genes can be identified. Accordingly, the presentinvention encompasses screening methods for genes encoding enzymesinvolved in the production of compounds with a particular nutritionalvalue and/or agronomic traits.

Further Applications of the System in Plants and Yeasts Use of System inBiofuel Production

The term “biofuel” as used herein is an alternative fuel made from plantand plant-derived resources. Renewable biofuels can be extracted fromorganic matter whose energy has been obtained through a process ofcarbon fixation or are made through the use or conversion of biomass.This biomass can be used directly for biofuels or can be converted toconvenient energy containing substances by thermal conversion, chemicalconversion, and biochemical conversion. This biomass conversion canresult in fuel in solid, liquid, or gas form. There are two types ofbiofuels: bioethanol and biodiesel. Bioethanol is mainly produced by thesugar fermentation process of cellulose (starch), which is mostlyderived from maize and sugar cane. Biodiesel on the other hand is mainlyproduced from oil crops such as rapeseed, palm, and soybean. Biofuelsare used mainly for transportation.

Enhancing Plant Properties for Biofuel Production

In particular embodiments, the methods using the System as describedherein are used to alter the properties of the cell wall in order tofacilitate access by key hydrolysing agents for a more efficient releaseof sugars for fermentation. In particular embodiments, the biosynthesisof cellulose and/or lignin are modified. Cellulose is the majorcomponent of the cell wall. The biosynthesis of cellulose and lignin areco-regulated. By reducing the proportion of lignin in a plant theproportion of cellulose can be increased. In particular embodiments, themethods described herein are used to downregulate lignin biosynthesis inthe plant so as to increase fermentable carbohydrates. Moreparticularly, the methods described herein are used to downregulate atleast a first lignin biosynthesis gene selected from the groupconsisting of 4-coumarate 3-hydroxylase (C3H), phenylalanineammonia-lyase (PAL), cinnamate 4-hydroxylase (C4H), hydroxycinnamoyltransferase (HCT), caffeic acid O-methyltransferase (COMT), caffeoyl CoA3-O-methyltransferase (CCoAOMT), ferulate 5-hydroxylase (F5H), cinnamylalcohol dehydrogenase (CAD), cinnamoyl CoA-reductase (CCR),4-coumarate-CoA ligase (4CL), monolignol-lignin-specificglycosyltransferase, and aldehyde dehydrogenase (ALDH) as disclosed inWO 2008064289 A2.

In particular embodiments, the methods described herein are used toproduce plant mass that produces lower levels of acetic acid duringfermentation (see also WO 2010096488). More particularly, the methodsdisclosed herein are used to generate mutations in homologs to Cas1L toreduce polysaccharide acetylation.

Modifying Yeast for Biofuel Production

In particular embodiments, the System provided herein is used forbioethanol production by recombinant micro-organisms. For instance, theSystem can be used to engineer micro-organisms, such as yeast, togenerate biofuel or biopolymers from fermentable sugars and optionallyto be able to degrade plant-derived lignocellulose derived fromagricultural waste as a source of fermentable sugars. In someembodiments, the System is used to modify endogenous metabolic pathwayswhich compete with the biofuel production pathway.

Accordingly, in more particular embodiments, the methods describedherein are used to modify a micro-organism as follows: to modify atleast one nucleic acid encoding for an enzyme in a metabolic pathway insaid host cell, wherein said pathway produces a metabolite other thanacetaldehyde from pyruvate or ethanol from acetaldehyde, and whereinsaid modification results in a reduced production of said metabolite, orto introduce at least one nucleic acid encoding for an inhibitor of saidenzyme.

Modifying Algae and Plants for Production of Vegetable Oils or Biofuels

Transgenic algae or other plants such as rape may be particularly usefulin the production of vegetable oils or biofuels such as alcohols(especially methanol and ethanol), for instance. These may be engineeredto express or overexpress high levels of oil or alcohols for use in theoil or biofuel industries.

According to particular embodiments of the invention, the System is usedto generate lipid-rich diatoms which are useful in biofuel production.

In particular embodiments it is envisaged to specifically modify genesthat are involved in the modification of the quantity of lipids and/orthe quality of the lipids produced by the algal cell. Examples of genesencoding enzymes involved in the pathways of fatty acid synthesis canencode proteins having for instance acetyl-CoA carboxylase, fatty acidsynthase, 3-ketoacyl_acyl-carrier protein synthase III,glycerol-3-phospate deshydrogenase (G3PDH), Enoyl-acyl carrier proteinreductase (Enoyl-ACP-reductase), glycerol-3-phosphate acyltransferase,lysophosphatidic acyl transferase or diacylglycerol acyltransferase,phospholipid:diacylglycerol acyltransferase, phoshatidate phosphatase,fatty acid thioesterase such as palmitoyi protein thioesterase, or malicenzyme activities. In further embodiments it is envisaged to generatediatoms that have increased lipid accumulation. This can be achieved bytargeting genes that decrease lipid catabolisation. Of particularinterest for use in the methods of the present invention are genesinvolved in the activation of both triacylglycerol and free fatty acids,as well as genes directly involved in β-oxidation of fatty acids, suchas acyl-CoA synthetase, 3-ketoacyl-CoA thiolase, acyl-CoA oxidaseactivity and phosphoglucomutase. The System and methods described hereincan be used to specifically activate such genes in diatoms as toincrease their lipid content.

Organisms such as microalgae are widely used for synthetic biology.Stovicek et al. (Metab. Eng. Comm., 2015; 2:13 describes genome editingof industrial yeast, for example, Saccharomyces cerevisae, toefficiently produce robust strains for industrial production. Stovicekused a CRISPR-Cas9 system codon-optimized for yeast to simultaneouslydisrupt both alleles of an endogenous gene and knock in a heterologousgene. Cas9 and guide RNA were expressed from genomic or episomal2μ-based vector locations. The authors also showed that gene disruptionefficiency could be improved by optimization of the levels of Cas9 andguide RNA expression. Hlavova et al. (Biotechnol. Adv. 2015) discussesdevelopment of species or strains of microalgae using techniques such asCRISPR to target nuclear and chloroplast genes for insertionalmutagenesis and screening.

U.S. Pat. No. 8,945,839 describes a method for engineering Micro-Algae(Chlamydomonas reinhardtii cells) species) using Cas9. Using similartools, the methods of the System described herein can be applied onChlamydomonas species and other algae. In particular embodiments, aCRISPR-Cas protein (e.g., Cas13), adenosine deaminase (which may befused to the CRISPR-Cas protein or an aptamer-binding adaptor protein),and guide RNA are introduced in algae expressed using a vector thatexpresses the CRISPR-Cas protein and optionally the adenosine deaminaseunder the control of a constitutive promoter such as Hsp70A-Rbc S2 orBeta2-tubulin. Guide RNA will be delivered using a vector containing T7promoter. Alternatively, mRNA and in vitro transcribed guide RNA can bedelivered to algal cells. Electroporation protocol follows standardrecommended protocol from the GeneArt Chlamydomonas Engineering kit.

The Use of System in the Generation of Micro-Organisms Capable of FattyAcid Production

In particular embodiments, the methods of the invention are used for thegeneration of genetically engineered micro-organisms capable of theproduction of fatty esters, such as fatty acid methyl esters (“FAME”)and fatty acid ethyl esters (“FAEE”),

Typically, host cells can be engineered to produce fatty esters from acarbon source, such as an alcohol, present in the medium, by expressionor overexpression of a gene encoding a thioesterase, a gene encoding anacyl-CoA synthase, and a gene encoding an ester synthase. Accordingly,the methods provided herein are used to modify a micro-organisms so asto overexpress or introduce a thioesterase gene, a gene encloding anacyl-CoA synthase, and a gene encoding an ester synthase. In particularembodiments, the thioesterase gene is selected from tesA, ‘tesA,tesB,fatB, fatB2,fatB3,fatA1, or fatA. In particular embodiments, thegene encoding an acyl-CoA synthase is selected from fadDJadK, BH3103,pf1-4354, EAV15023, fadD1, fadD2, RPC_4074,fadDD35, fadDD22, faa39, oran identified gene encoding an enzyme having the same properties. Inparticular embodiments, the gene encoding an ester synthase is a geneencoding a synthase/acyl-CoA:diacylglycerl acyltransferase fromSimmondsia chinensis, Acinetobacter sp. ADP, Alcanivorax borkumensis,Pseudomonas aeruginosa, Fundibacter jadensis, Arabidopsis thaliana, orAlkaligenes eutrophus, or a variant thereof. Additionally oralternatively, the methods provided herein are used to decreaseexpression in said micro-organism of of at least one of a gene encodingan acyl-CoA dehydrogenase, a gene encoding an outer membrane proteinreceptor, and a gene encoding a transcriptional regulator of fatty acidbiosynthesis. In particular embodiments one or more of these genes isinactivated, such as by introduction of a mutation. In particularembodiments, the gene encoding an acyl-CoA dehydrogenase is fadE. Inparticular embodiments, the gene encoding a transcriptional regulator offatty acid biosynthesis encodes a DNA transcription repressor, forexample, fabR.

Additionally or alternatively, said micro-organism is modified to reduceexpression of at least one of a gene encoding a pyruvate formate lyase,a gene encoding a lactate dehydrogenase, or both. In particularembodiments, the gene encoding a pyruvate formate lyase is pf1B. Inparticular embodiments, the gene encoding a lactate dehydrogenase isIdhA. In particular embodiments one or more of these genes isinactivated, such as by introduction of a mutation therein.

In particular embodiments, the micro-organism is selected from the genusEscherichia, Bacillus, Lactobacillus, Rhodococcus, Synechococcus,Synechoystis, Pseudomonas, Aspergillus, Trichoderma, Neurospora,Fusarium, Humicola, Rhizomucor, Kluyveromyces, Pichia, Mucor,Myceliophtora, Penicillium, Phanerochaete, Pleurotus, Trametes,Chrysosporium, Saccharomyces, Stenotrophamonas, Schizosaccharomyces,Yarrowia, or Streptomyces.

The Use of System in the Generation of Micro-Organisms Capable ofOrganic Acid Production

The methods provided herein are further used to engineer micro-organismscapable of organic acid production, more particularly from pentose orhexose sugars. In particular embodiments, the methods compriseintroducing into a micro-organism an exogenous LDH gene. In particularembodiments, the organic acid production in said micro-organisms isadditionally or alternatively increased by inactivating endogenous genesencoding proteins involved in an endogenous metabolic pathway whichproduces a metabolite other than the organic acid of interest and/orwherein the endogenous metabolic pathway consumes the organic acid. Inparticular embodiments, the modification ensures that the production ofthe metabolite other than the organic acid of interest is reduced.According to particular embodiments, the methods are used to introduceat least one engineered gene deletion and/or inactivation of anendogenous pathway in which the organic acid is consumed or a geneencoding a product involved in an endogenous pathway which produces ametabolite other than the organic acid of interest. In particularembodiments, the at least one engineered gene deletion or inactivationis in one or more gene encoding an enzyme selected from the groupconsisting of pyruvate decarboxylase (pdc), fumarate reductase, alcoholdehydrogenase (adh), acetaldehyde dehydrogenase, phosphoenolpyruvatecarboxylase (ppc), D-lactate dehydrogenase (d-1dh), L-lactatedehydrogenase (1-1dh), lactate 2-monooxygenase. In further embodimentsthe at least one engineered gene deletion and/or inactivation is in anendogenous gene encoding pyruvate decarboxylase (pdc).

In further embodiments, the micro-organism is engineered to producelactic acid and the at least one engineered gene deletion and/orinactivation is in an endogenous gene encoding lactate dehydrogenase.Additionally or alternatively, the micro-organism comprises at least oneengineered gene deletion or inactivation of an endogenous gene encodinga cytochrome-dependent lactate dehydrogenase, such as a cytochromeB2-dependent L-lactate dehydrogenase.

The Use of System in the Generation of Improved Xylose or CellobioseUtilizing Yeasts Strains

In particular embodiments, the System may be applied to select forimproved xylose or cellobiose utilizing yeast strains. Error-prone PCRcan be used to amplify one (or more) genes involved in the xyloseutilization or cellobiose utilization pathways. Examples of genesinvolved in xylose utilization pathways and cellobiose utilizationpathways may include, without limitation, those described in Ha, S. J.,et al. (2011) Proc. Natl. Acad. Sci. USA 108(2):504-9 and Galazka, J.M., et al. (2010) Science 330(6000):84-6. Resulting libraries ofdouble-stranded DNA molecules, each comprising a random mutation in sucha selected gene could be co-transformed with the components of theSystem into a yeast strain (for instance S288C) and strains can beselected with enhanced xylose or cellobiose utilization capacity, asdescribed in WO2015138855.

The Use of System in the Generation of Improved Yeasts Strains for Usein Isoprenoid Biosynthesis

Tadas Jakočiūnas et al. described the successful application of amultiplex CRISPR-Cas9 system for genome engineering of up to 5 differentgenomic loci in one transformation step in baker's yeast Saccharomycescerevisiae (Metabolic Engineering Volume 28, March 2015, Pages 213-222)resulting in strains with high mevalonate production, a key intermediatefor the industrially important isoprenoid biosynthesis pathway. Inparticular embodiments, the System may be applied in a multiplex genomeengineering method as described herein for identifying additional highproducing yeast strains for use in isoprenoid synthesis.

Improved Plants and Yeast Cells

The present invention also provides plants and yeast cells obtainableand obtained by the methods provided herein. The improved plantsobtained by the methods described herein may be useful in food or feedproduction through expression of genes which, for instance ensuretolerance to plant pests, herbicides, drought, low or high temperatures,excessive water, etc.

The improved plants obtained by the methods described herein, especiallycrops and algae may be useful in food or feed production throughexpression of, for instance, higher protein, carbohydrate, nutrient orvitamin levels than would normally be seen in the wildtype. In thisregard, improved plants, especially pulses and tubers are preferred.

Improved algae or other plants such as rape may be particularly usefulin the production of vegetable oils or biofuels such as alcohols(especially methanol and ethanol), for instance. These may be engineeredto express or overexpress high levels of oil or alcohols for use in theoil or biofuel industries.

The invention also provides for improved parts of a plant. Plant partsinclude, but are not limited to, leaves, stems, roots, tubers, seeds,endosperm, ovule, and pollen. Plant parts as envisaged herein may beviable, nonviable, regeneratable, and/or non-regeneratable.

It is also encompassed herein to provide plant cells and plantsgenerated according to the methods of the invention. Gametes, seeds,embryos, either zygotic or somatic, progeny or hybrids of plantscomprising the genetic modification, which are produced by traditionalbreeding methods, are also included within the scope of the presentinvention. Such plants may contain a heterologous or foreign DNAsequence inserted at or instead of a target sequence. Alternatively,such plants may contain only an alteration (mutation, deletion,insertion, substitution) in one or more nucleotides. As such, suchplants will only be different from their progenitor plants by thepresence of the particular modification.

Thus, the invention provides a plant, animal or cell, produced by thepresent methods, or a progeny thereof. The progeny may be a clone of theproduced plant or animal, or may result from sexual reproduction bycrossing with other individuals of the same species to introgressfurther desirable traits into their offspring. The cell may be in vivoor ex vivo in the cases of multicellular organisms, particularly animalsor plants.

The methods for genome editing using the System as described herein canbe used to confer desired traits on essentially any plant, algae,fungus, yeast, etc. A wide variety of plants, algae, fungus, yeast, etcand plant algae, fungus, yeast cell or tissue systems may be engineeredfor the desired physiological and agronomic characteristics describedherein using the nucleic acid constructs of the present disclosure andthe various transformation methods mentioned above.

In particular embodiments, the methods described herein are used tomodify endogenous genes or to modify their expression without thepermanent introduction into the genome of the plant, algae, fungus,yeast, etc of any foreign gene, including those encoding CRISPRcomponents, so as to avoid the presence of foreign DNA in the genome ofthe plant. This can be of interest as the regulatory requirements fornon-transgenic plants are less rigorous.

The methods described herein generally result in the generation of“improved plants, algae, fungi, yeast, etc” in that they have one ormore desirable traits compared to the wildtype plant. In particularembodiments, non-transgenic genetically modified plants, algae, fungi,yeast, etc., parts or cells are obtained, in that no exogenous DNAsequence is incorporated into the genome of any of the cells of theplant. In such embodiments, the improved plants, algae, fungi, yeast,etc. are non-transgenic. Where only the modification of an endogenousgene is ensured and no foreign genes are introduced or maintained in theplant, algae, fungi, yeast, etc. genome, the resulting geneticallymodified crops contain no foreign genes and can thus basically beconsidered non-transgenic. The different applications of the System forplant, algae, fungi, yeast, etc. genome editing include, but are notlimited to: editing of endogenous genes to confer an agricultural traitof interest. Examplary genes conferring agronomic traits include, butare not limited to genes that confer resistance to pests or diseases;genes involved in plant diseases, such as those listed in WO 2013046247;genes that confer resistance to herbicides, fungicides, or the like;genes involved in (abiotic) stress tolerance. Other aspects of the useof the CRISPR-Cas system include, but are not limited to: create (male)sterile plants; increasing the fertility stage in plants/algae etc;generate genetic variation in a crop of interest; affect fruit-ripening;increasing storage life of plants/algae etc; reducing allergen inplants/algae etc; ensure a value added trait (e.g. nutritionalimprovement); Sscreening methods for endogenous genes of interest;biofuel, fatty acid, organic acid, etc production.

System Can Be Used In Non-Human Organisms

In an aspect, the invention provides a non-human eukaryotic organism;preferably a multicellular eukaryotic organism, comprising a eukaryotichost cell according to any of the described embodiments. In otheraspects, the invention provides a eukaryotic organism; preferably amulticellular eukaryotic organism, comprising a eukaryotic host cellaccording to any of the described embodiments. The organism in someembodiments of these aspects may be an animal; for example a mammal.Also, the organism may be an arthropod such as an insect. The presentinvention may also be extended to other agricultural applications suchas, for example, farm and production animals. For example, pigs havemany features that make them attractive as biomedical models, especiallyin regenerative medicine. In particular, pigs with severe combinedimmunodeficiency (SCID) may provide useful models for regenerativemedicine, xenotransplantation (discussed also elsewhere herein), andtumor development and will aid in developing therapies for human SCIDpatients. Lee et al., (Proc Natl Acad Sci U S A. 2014 May 20;111(20):7260-5) utilized a reporter-guided transcription activator-likeeffector nuclease (TALEN) system to generated targeted modifications ofrecombination activating gene (RAG) 2 in somatic cells at highefficiency, including some that affected both alleles. The System may beapplied to a similar system.

The methods of Lee et al., (Proc Natl Acad Sci U S A. 2014 May 20;111(20):7260-5) may be applied to the present invention analogously asfollows. Mutated pigs are produced by targeted modification of RAG2 infetal fibroblast cells followed by SCNT and embryo transfer. Constructscoding for CRISPR Cas and a reporter are electroporated intofetal-derived fibroblast cells. After 48 h, transfected cells expressingthe green fluorescent protein are sorted into individual wells of a96-well plate at an estimated dilution of a single cell per well.Targeted modification of RAG2 are screened by amplifying a genomic DNAfragment flanking any CRISPR Cas cutting sites followed by sequencingthe PCR products. After screening and ensuring lack of off-sitemutations, cells carrying targeted modification of RAG2 are used forSCNT. The polar body, along with a portion of the adjacent cytoplasm ofoocyte, presumably containing the metaphase II plate, are removed, and adonor cell are placed in the perivitelline. The reconstructed embryosare then electrically porated to fuse the donor cell with the oocyte andthen chemically activated. The activated embryos are incubated inPorcine Zygote Medium 3 (PZM3) with 0.5 μM Scriptaid (S7817;Sigma-Aldrich) for 14-16 h. Embryos are then washed to remove theScriptaid and cultured in PZM3 until they were transferred into theoviducts of surrogate pigs.

The present invention is also applicable to modifying SNPs of otheranimals, such as cows. Tan et al. (Proc Natl Acad Sci U S A. 2013 Oct.8; 110(41): 16526-16531) expanded the livestock gene editing toolbox toinclude transcription activator-like (TAL) effector nuclease (TALEN)-and clustered regularly interspaced short palindromic repeats(CRISPR)/Cas9-stimulated homology-directed repair (HDR) using plasmid,rAAV, and oligonucleotide templates. Gene specific guide RNA sequenceswere cloned into the Church lab guide RNA vector (Addgene ID: 41824)according to their methods (Mali P, et al. (2013) RNA-Guided HumanGenome Engineering via Cas9. Science 339(6121):823-826). The Cas9nuclease was provided either by co-transfection of the hCas9 plasmid(Addgene ID: 41815) or mRNA synthesized from RCIScript-hCas9. ThisRCIScript-hCas9 was constructed by sub-cloning the XbaI-AgeI fragmentfrom the hCas9 plasmid (encompassing the hCas9 cDNA) into the RCIScriptplasmid.

Heo et al. (Stem Cells Dev. 2015 Feb. 1; 24(3):393-402. doi:10.1089/scd.2014.0278. Epub 2014 Nov. 3) reported highly efficient genetargeting in the bovine genome using bovine pluripotent cells andclustered regularly interspaced short palindromic repeat (CRISPR)/Cas9nuclease. First, Heo et al. generate induced pluripotent stem cells(iPSCs) from bovine somatic fibroblasts by the ectopic expression ofyamanaka factors and GSK3β and MEK inhibitor (2i) treatment. Heo et al.observed that these bovine iPSCs are highly similar to naive pluripotentstem cells with regard to gene expression and developmental potential interatomas. Moreover, CRISPR-Cas9 nuclease, which was specific for thebovine NANOG locus, showed highly efficient editing of the bovine genomein bovine iPSCs and embryos.

Igenity® provides a profile analysis of animals, such as cows, toperform and transmit traits of economic traits of economic importance,such as carcass composition, carcass quality, maternal and reproductivetraits and average daily gain. The analysis of a comprehensive Igenity®profile begins with the discovery of DNA markers (most often singlenucleotide polymorphisms or SNPs). All the markers behind the Igenity®profile were discovered by independent scientists at researchinstitutions, including universities, research organizations, andgovernment entities such as USDA. Markers are then analyzed at Igenity®in validation populations. Igenity® uses multiple resource populationsthat represent various production environments and biological types,often working with industry partners from the seedstock, cow-calf,feedlot and/or packing segments of the beef industry to collectphenotypes that are not commonly available. Cattle genome databases arewidely available, see, e.g., the NAGRP Cattle Genome CoordinationProgram (www.animalgenome.org/cattle/maps/db.html). Thus, the presentinvention maybe applied to target bovine SNPs. One of skill in the artmay utilize the above protocols for targeting SNPs and apply them tobovine SNPs as described, for example, by Tan et al. or Heo et al.

Qingjian Zou et al. (Journal of Molecular Cell Biology Advance Accesspublished Oct. 12, 2015) demonstrated increased muscle mass in dogs bytargeting targeting the first exon of the dog Myostatin (MSTN) gene (anegative regulator of skeletal muscle mass). First, the efficiency ofthe sgRNA was validated, using cotransfection of the the sgRNA targetingMSTN with a Cas9 vector into canine embryonic fibroblasts (CEFs).Thereafter, MSTN KO dogs were generated by micro-injecting embryos withnormal morphology with a mixture of Cas9 mRNA and MSTN sgRNA andauto-transplantation of the zygotes into the oviduct of the same femaledog. The knock-out puppies displayed an obvious muscular phenotype onthighs compared with its wild-type littermate sister. This can also beperformed using the Systems provided herein.

Livestock—Pigs

Viral targets in livestock may include, in some embodiments, porcineCD163, for example on porcine macrophages. CD163 is associated withinfection (thought to be through viral cell entry) by PRRSv (PorcineReproductive and Respiratory Syndrome virus, an arterivirus). Infectionby PRRSv, especially of porcine alveolar macrophages (found in thelung), results in a previously incurable porcine syndrome (“Mysteryswine disease” or “blue ear disease”) that causes suffering, includingreproductive failure, weight loss and high mortality rates in domesticpigs. Opportunistic infections, such as enzootic pneumonia, meningitisand ear oedema, are often seen due to immune deficiency through loss ofmacrophage activity. It also has significant economic and environmentalrepercussions due to increased antibiotic use and financial loss (anestimated $660 m per year).

As reported by Kristin M Whitworth and Dr Randall Prather et al. (NatureBiotech 3434 published online 7 Dec. 2015) at the University of Missouriand in collaboration with Genus Plc, CD163 was targeted usingCRISPR-Cas9 and the offspring of edited pigs were resistant when exposedto PRRSv. One founder male and one founder female, both of whom hadmutations in exon 7 of CD163, were bred to produce offspring. Thefounder male possessed an 11-bp deletion in exon 7 on one allele, whichresults in a frameshift mutation and missense translation at amino acid45 in domain 5 and a subsequent premature stop codon at amino acid 64.The other allele had a 2-bp addition in exon 7 and a 377-bp deletion inthe preceding intron, which were predicted to result in the expressionof the first 49 amino acids of domain 5, followed by a premature stopcode at amino acid 85. The sow had a 7 bp addition in one allele thatwhen translated was predicted to express the first 48 amino acids ofdomain 5, followed by a premature stop codon at amino acid 70. The sow'sother allele was unamplifiable. Selected offspring were predicted to bea null animal (CD163−/−), i.e. a CD163 knock out.

Accordingly, in some embodiments, porcine alveolar macrophages may betargeted by the CRISPR protein. In some embodiments, porcine CD163 maybe targeted by the CRISPR protein. In some embodiments, porcine CD163may be knocked out through induction of a DSB or through insertions ordeletions, for example targeting deletion or modification of exon 7,including one or more of those described above, or in other regions ofthe gene, for example deletion or modification of exon 5.

An edited pig and its progeny are also envisaged, for example a CD163knock out pig. This may be for livestock, breeding or modelling purposes(i.e. a porcine model). Semen comprising the gene knock out is alsoprovided.

CD163 is a member of the scavenger receptor cysteine-rich (SRCR)superfamily. Based on in vitro studies SRCR domain 5 of the protein isthe domain responsible for unpackaging and release of the viral genome.As such, other members of the SRCR superfamily may also be targeted inorder to assess resistance to other viruses. PRRSV is also a member ofthe mammalian arterivirus group, which also includes murine lactatedehydrogenase-elevating virus, simian hemorrhagic fever virus and equinearteritis virus. The arteriviruses share important pathogenesisproperties, including macrophage tropism and the capacity to cause bothsevere disease and persistent infection. Accordingly, arteriviruses, andin particular murine lactate dehydrogenase-elevating virus, simianhemorrhagic fever virus and equine arteritis virus, may be targeted, forexample through porcine CD163 or homologues thereof in other species,and murine, simian and equine models and knockout also provided.

Indeed, this approach may be extended to viruses or bacteria that causeother livestock diseases that may be transmitted to humans, such asSwine Influenza Virus (SIV) strains which include influenza C and thesubtypes of influenza A known as H1N1, H1N2, H2N1, H3N1, H3N2, and H2N3,as well as pneumonia, meningitis and oedema mentioned above.

In some embodiments, the System described herein can be used togenetically modify a pig genome to inactivate one or more porcineendogenous retrovirus (PERVs) loci to facilitate clinical application ofporcine-to-human xenotransplantation. See Yang et al., Science350(6264):1101-1104 (2015), which is incorporated herein by reference inits entirey. In some embodiments, the System described herein can beused to produce a genetically modified pig that does not comprise anyactive porcine endogenous retrovirus (PERVs) locus.

Therapeutic Targeting with System

As will be apparent, it is envisaged that System can be used to targetany polynucleotide sequence of interest. The invention provides anon-naturally occurring or engineered composition, or one or morepolynucleotides encoding components of said composition, or vector ordelivery systems comprising one or more polynucleotides encodingcomponents of said composition for use in a modifying a target cell invivo, ex vivo or in vitro and, may be conducted in a manner alters thecell such that once modified the progeny or cell line of the CRISPRmodified cell retains the altered phenotype. The modified cells andprogeny may be part of a multi-cellular organism such as a plant oranimal with ex vivo or in vivo application of CRISPR system to desiredcell types. The CRISPR invention may be a therapeutic method oftreatment. The therapeutic method of treatment may comprise gene orgenome editing, or gene therapy.

Adoptive Cell Therapies

The present invention also contemplates use of the System describedherein to modify cells for adoptive therapies. Aspects of the inventionaccordingly involve the adoptive transfer of immune system cells, suchas T cells, specific for selected antigens, such as tumor associatedantigens (see Maus et al., 2014, Adoptive Immunotherapy for Cancer orViruses, Annual Review of Immunology, Vol. 32: 189-225; Rosenberg andRestifo, 2015, Adoptive cell transfer as personalized immunotherapy forhuman cancer, Science Vol. 348 no. 6230 pp. 62-68; and, Restifo et al.,2015, Adoptive immunotherapy for cancer: harnessing the T cell response.Nat. Rev. Immunol. 12(4): 269-281; and Jenson and Riddell, 2014, Designand implementation of adoptive therapy with chimeric antigenreceptor-modified T cells. Immunol Rev. 257(1): 127-144). Variousstrategies may for example be employed to genetically modify T cells byaltering the specificity of the T cell receptor (TCR) for example byintroducing new TCR α and β chains with selected peptide specificity(see U.S. Pat. No. 8,697,854; PCT Patent Publications: WO2003020763,WO2004033685, WO2004044004, WO2005114215, WO2006000830, WO2008038002,WO2008039818, WO2004074322, WO2005113595, WO2006125962, WO2013166321,WO2013039889, WO2014018863, WO2014083173; U.S. Pat. No. 8,088,379).

As an alternative to, or addition to, TCR modifications, chimericantigen receptors (CARs) may be used in order to generateimmunoresponsive cells, such as T cells, specific for selected targets,such as malignant cells, with a wide variety of receptor chimeraconstructs having been described (see U.S. Pat. Nos. 5,843,728;5,851,828; 5,912,170; 6,004,811; 6,284,240; 6,392,013; 6,410,014;6,753,162; 8,211,422; and, PCT Publication WO9215322). Alternative CARconstructs may be characterized as belonging to successive generations.First-generation CARs typically consist of a single-chain variablefragment of an antibody specific for an antigen, for example comprisinga VL linked to a VH of a specific antibody, linked by a flexible linker,for example by a CD8α hinge domain and a CD8α transmembrane domain, tothe transmembrane and intracellular signaling domains of either CD3ζ orFcRγ (scFv-CD3ζ or scFv-FcRγ; see U.S. Pat. Nos. 7,741,465; 5,912,172;5,906,936). Second-generation CARs incorporate the intracellular domainsof one or more costimulatory molecules, such as CD28, OX40 (CD134), or4-1BB (CD137) within the endodomain (for examplescFv-CD28/OX40/4-1BB-CD3ζ; see U.S. Pat. Nos. 8,911,993; 8,916,381;8,975,071; 9,101,584; 9,102,760; 9,102,761). Third-generation CARsinclude a combination of costimulatory endodomains, such a CD3ζ-chain,CD97, GDI 1a-CD18, CD2, ICOS, CD27, CD154, CDS, OX40, 4-1BB, or CD28signaling domains (for example scFv-CD28-4-1BB-CD3ζ orscFv-CD28-OX40-CD3ζ; see U.S. Pat. Nos. 8,906,682; 8,399,645; 5,686,281;PCT Publication No. WO2014134165; PCT Publication No. WO2012079000).Alternatively, costimulation may be orchestrated by expressing CARs inantigen-specific T cells, chosen so as to be activated and expandedfollowing engagement of their native αβTCR, for example by antigen onprofessional antigen-presenting cells, with attendant costimulation. Inaddition, additional engineered receptors may be provided on theimmunoresponsive cells, for example to improve targeting of a T-cellattack and/or minimize side effects.

Alternative techniques may be used to transform target immunoresponsivecells, such as protoplast fusion, lipofection, transfection orelectroporation. A wide variety of vectors may be used, such asretroviral vectors, lentiviral vectors, adenoviral vectors,adeno-associated viral vectors, plasmids or transposons, such as aSleeping Beauty transposon (see U.S. Pat. Nos. 6,489,458; 7,148,203;7,160,682; 7,985,739; 8,227,432), may be used to introduce CARs, forexample using 2nd generation antigen-specific CARs signaling throughCD3t and either CD28 or CD137. Viral vectors may for example includevectors based on HIV, SV40, EBV, HSV or BPV.

Cells that are targeted for transformation may for example include Tcells, Natural Killer (NK) cells, cytotoxic T lymphocytes (CTL),regulatory T cells, human embryonic stem cells, tumor-infiltratinglymphocytes (TIL) or a pluripotent stem cell from which lymphoid cellsmay be differentiated. T cells expressing a desired CAR may for examplebe selected through co-culture with γ-irradiated activating andpropagating cells (AaPC), which co-express the cancer antigen andco-stimulatory molecules. The engineered CAR T-cells may be expanded,for example by co-culture on AaPC in presence of soluble factors, suchas IL-2 and IL-21. This expansion may for example be carried out so asto provide memory CAR+ T cells (which may for example be assayed bynon-enzymatic digital array and/or multi-panel flow cytometry). In thisway, CAR T cells may be provided that have specific cytotoxic activityagainst antigen-bearing tumors (optionally in conjunction withproduction of desired chemokines such as interferon-γ). CART cells ofthis kind may for example be used in animal models, for example tothreat tumor xenografts.

Approaches such as the foregoing may be adapted to provide methods oftreating and/or increasing survival of a subject having a disease, suchas a neoplasia, for example by administering an effective amount of animmunoresponsive cell comprising an antigen recognizing receptor thatbinds a selected antigen, wherein the binding activates theimmunoreponsive cell, thereby treating or preventing the disease (suchas a neoplasia, a pathogen infection, an autoimmune disorder, or anallogeneic transplant reaction). Dosing in CAR T cell therapies may forexample involve administration of from 106 to 109 cells/kg, with orwithout a course of lymphodepletion, for example with cyclophosphamide.

In one embodiment, the treatment can be administrated into patientsundergoing an immunosuppressive treatment. The cells or population ofcells, may be made resistant to at least one immunosuppressive agent dueto the inactivation of a gene encoding a receptor for suchimmunosuppressive agent. Not being bound by a theory, theimmunosuppressive treatment should help the selection and expansion ofthe immunoresponsive or T cells according to the invention within thepatient.

The administration of the cells or population of cells according to thepresent invention may be carried out in any convenient manner, includingby aerosol inhalation, injection, ingestion, transfusion, implantationor transplantation. The cells or population of cells may be administeredto a patient subcutaneously, intradermally, intratumorally,intranodally, intramedullary, intramuscularly, by intravenous orintralymphatic injection, or intraperitoneally. In one embodiment, thecell compositions of the present invention are preferably administeredby intravenous injection.

The administration of the cells or population of cells can consist ofthe administration of 104-109 cells per kg body weight, preferably 105to 106 cells/kg body weight including all integer values of cell numberswithin those ranges. Dosing in CAR T cell therapies may for exampleinvolve administration of from 106 to 109 cells/kg, with or without acourse of lymphodepletion, for example with cyclophosphamide. The cellsor population of cells can be administrated in one or more doses. Inanother embodiment, the effective amount of cells are administrated as asingle dose. In another embodiment, the effective amount of cells areadministrated as more than one dose over a period time. Timing ofadministration is within the judgment of managing physician and dependson the clinical condition of the patient. The cells or population ofcells may be obtained from any source, such as a blood bank or a donor.While individual needs vary, determination of optimal ranges ofeffective amounts of a given cell type for a particular disease orconditions are within the skill of one in the art. An effective amountmeans an amount which provides a therapeutic or prophylactic benefit.The dosage administrated will be dependent upon the age, health andweight of the recipient, kind of concurrent treatment, if any, frequencyof treatment and the nature of the effect desired.

In another embodiment, the effective amount of cells or compositioncomprising those cells are administrated parenterally. Theadministration can be an intravenous administration. The administrationcan be directly done by injection within a tumor.

To guard against possible adverse reactions, engineered immunoresponsivecells may be equipped with a transgenic safety switch, in the form of atransgene that renders the cells vulnerable to exposure to a specificsignal. For example, the herpes simplex viral thymidine kinase (TK) genemay be used in this way, for example by introduction into allogeneic Tlymphocytes used as donor lymphocyte infusions following stem celltransplantation (Greco, et al., Improving the safety of cell therapywith the TK-suicide gene. Front. Pharmacol. 2015; 6: 95). In such cells,administration of a nucleoside prodrug such as ganciclovir or acyclovircauses cell death. Alternative safety switch constructs includeinducible caspase 9, for example triggered by administration of asmall-molecule dimerizer that brings together two nonfunctional icasp9molecules to form the active enzyme. A wide variety of alternativeapproaches to implementing cellular proliferation controls have beendescribed (see U.S. Patent Publication No. 20130071414; PCT PatentPublication WO2011146862; PCT Patent Publication WO2014011987; PCTPatent Publication WO2013040371; Zhou et al. BLOOD, 2014,123/25:3895-3905; Di Stasi et al., The New England Journal of Medicine2011; 365:1673-1683; Sadelain M, The New England Journal of Medicine2011; 365:1735-173; Ramos et al., Stem Cells 28(6):1107-15 (2010)).

In a further refinement of adoptive therapies, genome editing with aAD-functionalized CRISPR-Cas system as described herein may be used totailor immunoresponsive cells to alternative implementations, forexample providing edited CAR T cells (see Poirot et al., 2015, Multiplexgenome edited T-cell manufacturing platform for “off-the-shelf” adoptiveT-cell immunotherapies, Cancer Res 75 (18): 3853). For example,immunoresponsive cells may be edited to delete expression of some or allof the class of HLA type II and/or type I molecules, or to knockoutselected genes that may inhibit the desired immune response, such as thePD1 gene.

Cells may be edited using a System as described herein. Systems may bedelivered to an immune cell by any method described herein. In preferredembodiments, cells are edited ex vivo and transferred to a subject inneed thereof. Immunoresponsive cells, CAR-T cells or any cells used foradoptive cell transfer may be edited. Editing may be performed toeliminate potential alloreactive T-cell receptors (TCR), disrupt thetarget of a chemotherapeutic agent, block an immune checkpoint, activatea T cell, and/or increase the differentiation and/or proliferation offunctionally exhausted or dysfunctional CD8+ T-cells (see PCT PatentPublications: WO2013176915, WO2014059173, WO2014172606, WO2014184744,and WO2014191128). Editing may result in inactivation of a gene.

T cell receptors (TCR) are cell surface receptors that participate inthe activation of T cells in response to the presentation of antigen.The TCR is generally made from two chains, α and β, which assemble toform a heterodimer and associates with the CD3-transducing subunits toform the T cell receptor complex present on the cell surface. Each α andβ chain of the TCR consists of an immunoglobulin-like N-terminalvariable (V) and constant (C) region, a hydrophobic transmembranedomain, and a short cytoplasmic region. As for immunoglobulin molecules,the variable region of the α and β chains are generated by V(D)Jrecombination, creating a large diversity of antigen specificitieswithin the population of T cells. However, in contrast toimmunoglobulins that recognize intact antigen, T cells are activated byprocessed peptide fragments in association with an MHC molecule,introducing an extra dimension to antigen recognition by T cells, knownas MHC restriction. Recognition of MHC disparities between the donor andrecipient through the T cell receptor leads to T cell proliferation andthe potential development of graft versus host disease (GVHD). Theinactivation of TCRα or TCRβ can result in the elimination of the TCRfrom the surface of T cells preventing recognition of alloantigen andthus GVHD. However, TCR disruption generally results in the eliminationof the CD3 signaling component and alters the means of further T cellexpansion.

Allogeneic cells are rapidly rejected by the host immune system. It hasbeen demonstrated that, allogeneic leukocytes present in non-irradiatedblood products will persist for no more than 5 to 6 days (Boni, Muranskiet al. 2008 Blood 1; 112(12):4746-54). Thus, to prevent rejection ofallogeneic cells, the host's immune system usually has to be suppressedto some extent. However, in the case of adoptive cell transfer the useof immunosuppressive drugs also have a detrimental effect on theintroduced therapeutic T cells. Therefore, to effectively use anadoptive immunotherapy approach in these conditions, the introducedcells would need to be resistant to the immunosuppressive treatment.Thus, in a particular embodiment, the present invention furthercomprises a step of modifying T cells to make them resistant to animmunosuppressive agent, preferably by inactivating at least one geneencoding a target for an immunosuppressive agent. An immunosuppressiveagent is an agent that suppresses immune function by one of severalmechanisms of action. An immunosuppressive agent can be, but is notlimited to a calcineurin inhibitor, a target of rapamycin, aninterleukin-2 receptor α-chain blocker, an inhibitor of inosinemonophosphate dehydrogenase, an inhibitor of dihydrofolic acidreductase, a corticosteroid or an immunosuppressive antimetabolite. Thepresent invention allows conferring immunosuppressive resistance to Tcells for immunotherapy by inactivating the target of theimmunosuppressive agent in T cells. As non-limiting examples, targetsfor an immunosuppressive agent can be a receptor for animmunosuppressive agent such as: CD52, glucocorticoid receptor (GR), aFKBP family gene member and a cyclophilin family gene member.

Immune checkpoints are inhibitory pathways that slow down or stop immunereactions and prevent excessive tissue damage from uncontrolled activityof immune cells. In certain embodiments, the immune checkpoint targetedis the programmed death-1 (PD-1 or CD279) gene (PDCD1). In otherembodiments, the immune checkpoint targeted is cytotoxicT-lymphocyte-associated antigen (CTLA-4). In additional embodiments, theimmune checkpoint targeted is another member of the CD28 and CTLA4 Igsuperfamily such as BTLA, LAG3, ICOS, PDL1 or KIR. In further additionalembodiments, the immune checkpoint targeted is a member of the TNFRsuperfamily such as CD40, OX40, CD137, GITR, CD27 or TIM-3.

Additional immune checkpoints include Src homology 2 domain-containingprotein tyrosine phosphatase 1 (SHP-1) (Watson H A, et al., SHP-1: thenext checkpoint target for cancer immunotherapy? Biochem Soc Trans. 2016Apr. 15; 44(2):356-62). SHP-1 is a widely expressed inhibitory proteintyrosine phosphatase (PTP). In T-cells, it is a negative regulator ofantigen-dependent activation and proliferation. It is a cytosolicprotein, and therefore not amenable to antibody-mediated therapies, butits role in activation and proliferation makes it an attractive targetfor genetic manipulation in adoptive transfer strategies, such aschimeric antigen receptor (CAR) T cells. Immune checkpoints may alsoinclude T cell immunoreceptor with Ig and ITIM domains(TIGIT/Vstm3/WUCAM/VSIG9) and VISTA (Le Mercier I, et al., (2015) BeyondCTLA-4 and PD-1, the generation Z of negative checkpoint regulators.Front. Immunol. 6:418).

WO2014172606 relates to the use of MT1 and/or MT1 inhibitors to increaseproliferation and/or activity of exhausted CD8+ T-cells and to decreaseCD8+ T-cell exhaustion (e.g., decrease functionally exhausted orunresponsive CD8+ immune cells). In certain embodiments,metallothioneins are targeted by gene editing in adoptively transferredT cells.

In certain embodiments, targets of gene editing may be at least onetargeted locus involved in the expression of an immune checkpointprotein. Such targets may include, but are not limited to CTLA4, PPP2CA,PPP2CB, PTPN6, PTPN22, PDCD1, ICOS (CD278), PDL1, KIR, LAG3, HAVCR2,BTLA, CD160, TIGIT, CD96, CRTAM, LAIR1, SIGLEC7, SIGLEC9, CD244 (2B4),TNFRSF10B, TNFRSF10A, CASP8, CASP10, CASP3, CASP6, CASP7, FADD, FAS,TGFBRII, TGFRBRI, SMAD2, SMAD3, SMAD4, SMAD10, SKI, SKIL, TGIF1, IL10RA,IL10RB, HMOX2, IL6R, IL6ST, EIF2AK4, CSK, PAG1, SIT1, FOXP3, PRDM1,BATF, VISTA, GUCY1A2, GUCY1A3, GUCY1B2, GUCY1B3, MT1, MT2, CD40, OX40,CD137, GITR, CD27, SHP-1 or TIM-3. In preferred embodiments, the genelocus involved in the expression of PD-1 or CTLA-4 genes is targeted. Inother preferred embodiments, combinations of genes are targeted, such asbut not limited to PD-1 and TIGIT.

In other embodiments, at least two genes are edited. Pairs of genes mayinclude, but are not limited to PD1 and TCRα, PD1 and TCRβ, CTLA-4 andTCRα, CTLA-4 and TCRβ, LAG3 and TCRα, LAG3 and TCRβ, Tim3 and TCRα, Tim3and TCRβ, BTLA and TCRα, BTLA and TCRβ, BY55 and TCRα, BY55 and TCRβ,TIGIT and TCRα, TIGIT and TCRβ, B7H5 and TCRα, B7H5 and TCRβ, LAIR1 andTCRα, LAIR1 and TCRβ, SIGLEC10 and TCRα, SIGLEC10 and TCRβ, 2B4 andTCRα, 2B4 and TCRβ.

Whether prior to or after genetic modification of the T cells, the Tcells can be activated and expanded generally using methods asdescribed, for example, in U.S. Pat. Nos. 6,352,694; 6,534,055;6,905,680; 5,858,358; 6,887,466; 6,905,681; 7,144,575; 7,232,566;7,175,843; 5,883,223; 6,905,874; 6,797,514; 6,867,041; and 7,572,631. Tcells can be expanded in vitro or in vivo.

The practice of the present invention employs techniques known in thefield of immunology, biochemistry, chemistry, molecular biology,microbiology, cell biology, genomics and recombinant DNA, which arewithin the skill of the art. See MOLECULAR CLONING: A LABORATORY MANUAL,2nd edition (1989) (Sambrook, Fritsch and Maniatis); MOLECULAR CLONING:A LABORATORY MANUAL, 4th edition (2012) (Green and Sambrook); CURRENTPROTOCOLS IN MOLECULAR BIOLOGY (1987) (F. M. Ausubel, et al. eds.); theseries METHODS IN ENZYMOLOGY (Academic Press, Inc.); PCR 2: A PRACTICALAPPROACH (1995) (M. J. MacPherson, B. D. Hames and G. R. Taylor eds.);ANTIBODIES, A LABORATORY MANUAL (1988) (Harlow and Lane, eds.);ANTIBODIES A LABORATORY MANUAL, 2nd edition (2013) (E. A. Greenfielded.); and ANIMAL CELL CULTURE (1987) (R.I. Freshney, ed.).

Correction of Disease-Associated Mutations and Pathogenic SNPs

In one aspect, the invention described herein provides methods formodifying an adenosine residue at a target locus with the aim ofremedying and/or preventing a diseased condition that is or is likely tobe caused by a G-to-A or C-to-T point mutation or a pathogenic singlenucleotide polymorphism (SNP).

Diseases Affecting the Brain and Central Nervous System

Pathogenic G-to-A or C-to-T mutations/SNPs associated with variousdiseases affecting the brain and central nervous system are reported inthe ClinVar database and disclosed in Table A, including but not limitedto Alzheimer's Disease, Parkinson's Disease, Autism, Amyotrophyiclateral sclerosis (ALS), Schizophrenia, Adrenoleukodystrophy, AicardiGoutieres syndrome, Fabry disease, Lesch-Nyhan syndrome, and MenkesDisease. Accordingly, an aspect of the invention relates to a method forcorrecting one or more pathogenic G-to-A or C-to-T mutations/SNPsassociated with any of these diseases, as discussed below.

Alzheimer's Disease

In some embodiments, the methods, systems, and compositions describedherein are used to correct one or more pathogenic G-to-A or C-to-Tmutations/SNPs associated with Alzheimer's Disease. In some embodiments,the pathogenic mutations/SNPs are present in at least one gene selectedfrom PSEN1, PSEN2, and APP, including at least the followings:

-   NM_000021.3(PSEN1):c.796G>A (p.Gly266Ser)-   NM_000484.3(APP):c.2017G>A (p.Ala673Thr)-   NM_000484.3(APP):c.2149G>A (p.Val717Ile)-   NM_000484.3(APP):c.2137G>A (p.Ala713Thr)-   NM_000484.3(APP):c.2143G>A (p.Val715Met)-   NM_000484.3(APP):c.2141C>T (p.Thr714Ile)-   NM_000021.3(PSEN1):c.438G>A (p.Met146Ile)-   NM_000021.3(PSEN1):c.1229G>A (p.Cys410Tyr)-   NM_000021.3(PSEN1):c.487C>T (p.His163Tyr)-   NM_000021.3(PSEN1):c.799C>T (p.Pro267Ser)-   NM_000021.3(PSEN1):c.236C>T (p.Ala79Val)-   NM_000021.3(PSEN1):c.509C>T (p.Ser170Phe)-   NM_000447.2(PSEN2):c.1289C>T (p.Thr430Met)-   NM_000447.2(PSEN2):c.717G>A (p.Met239Ile)-   NM_000447.2(PSEN2):c.254C>T (p.Ala85Val)-   NM_000021.3(PSEN1):c.806G>A (p.Arg269His)-   NM_000484.3(APP):c.2018C>T (p.Ala673Val).    See Table A. Accordingly, an aspect of the invention relates to a    method for treating or preventing Alzheimer's Disease by correcting    one or more pathogenic G-to-A or C-to-T mutations/SNPs, particularly    one or more pathogenic G-to-A or C-to-T mutations/SNPs present in at    least one gene selected from PSEN1, PSEN2, and APP, and more    particularly one or more pathogenic G-to-A or C-to-T mutations/SNPs    described above.

Parkinson's Disease

In some embodiments, the methods, systems, and compositions describedherein are used to correct one or more pathogenic G-to-A or C-to-Tmutations/SNPs associated with Parkinson's Disease. In some embodiments,In some embodiment, the pathogenic mutations/SNPs are present in atleast one gene selected from SNCA, PLA2G6, FBXO7, VPS35, EIF4G1, DNAJC6,PRKN, SYNJ1, CHCHD2, PINK1, PARK7, LRRK2, ATP13A2, and GBA, including atleast the followings:

-   NM_000345.3(SNCA):c.157G>A (p.Ala53Thr)-   NM_000345.3(SNCA):c.152G>A (p.Gly5lAsp)-   NM_003560.3(PLA2G6):c.2222G>A (p.Arg741Gln)-   NM_003560.3(PLA2G6):c.2239C>T (p.Arg747Trp)-   NM_003560.3(PLA2G6):c.1904G>A (p.Arg635Gln)-   NM_003560.3(PLA2G6):c.1354C>T (p.Gln452Ter)-   NM_012179.3(FBX07):c.1492C>T (p.Arg498Ter)-   NM_012179.3(FBX07):c.65C>T (p.Thr22Met)-   NM_018206.5(VPS35):c.1858G>A (p.Asp620Asn)-   NM_198241.2(EIF4G1):c.3614G>A (p.Arg1205His)-   NM_198241.2(EIF4G1):c.1505C>T (p.Ala502Val)-   NM_001256865.1(DNAJC6):c.2200C>T (p.Gln734Ter)-   NM_001256865.1(DNAJC6):c.2326C>T (p.Gln776Ter)-   NM_004562.2(PRKN):c.931C>T (p.Gln311Ter)-   NM_004562.2(PRKN):c.1358G>A (p.Trp453Ter)-   NM_004562.2(PRKN):c.635G>A (p.Cys212Tyr)-   NM_203446.2(SYNJ1):c.773G>A (p.Arg258Gln)-   NM_001320327.1(CHCHD2):c.182C>T (p.Thr61Ile)-   NM_001320327.1(CHCHD2):c.434G>A (p.Arg145Gln)-   NM_001320327.1(CHCHD2):c.300+5G>A-   NM_032409.2(PINK1):c.926G>A (p.Gly309Asp)-   NM_032409.2(PINK1):c.1311G>A (p.Trp437Ter)-   NM_032409.2(PINK1):c.736C>T (p.Arg246Ter)-   NM_032409.2(PINK1):c.836G>A (p.Arg279His)-   NM_032409.2(PINK1):c.938C>T (p.Thr313Met)-   NM_032409.2(PINK1):c.1366C>T (p.Gln456Ter)-   NM_007262.4(PARK7):c.78G>A (p.Met26Ile)-   NM_198578.3(LRRK2):c.4321C>T (p.Arg1441Cys)-   NM_198578.3(LRRK2):c.4322G>A (p.Arg1441His)-   NM_198578.3(LRRK2):c.1256C>T (p.Ala419Val)-   NM_198578.3(LRRK2):c.6055G>A (p.Gly2019Ser)-   NM_022089.3(ATP13A2):c.1306+5G>A-   NM_022089.3(ATP13A2):c.2629G>A (p.Gly877Arg)-   NM_022089.3(ATP13A2):c.490C>T (p.Arg164Trp)-   NM_001005741.2(GBA):c.1444G>A (p.Asp482Asn)-   m.15950G>A.    See Table A. Accordingly, an aspect of the invention relates to a    method for treating or preventing Parkinson's Disease by correcting    one or more pathogenic G-to-A or C-to-T mutations/SNPs, particularly    one or more pathogenic G-to-A or C-to-T mutations/SNPs in at least    one gene selected from SNCA, PLA2G6, FBXO7, VPS35, EIF4G1, DNAJC6,    PRKN, SYNJ1, CHCHD2, PINK1, PARK7, LRRK2, ATP13A2, and GBA, and more    particularly one or more pathogenic G-to-A or C-to-T mutations/SNPs    described above.

Autism

In some embodiments, the methods, systems, and compositions describedherein are used to correct one or more pathogenic G-to-A or C-to-Tmutations/SNPs associated with Autism. In some embodiments, thepathogenic mutations/SNPs are present in at least one gene selected fromMECP2, NLGN3, SLC9A9, EHMT1, CHD8, NLGN4X , GSPT2, and PTEN, includingat least the followings:

-   NM_001110792.1(MECP2):c.916C>T (p.Arg306Ter)-   NM_004992.3(MECP2):c.473C>T (p.Thr158Met)-   NM_018977.3(NLGN3):c.1351C>T (p.Arg451Cys)-   NM_173653.3(SLC9A9):c.1267C>T (p.Arg423Ter)-   NM_024757.4(EHMT1):c.3413G>A (p.Trp1138Ter)-   NM_020920.3(CHD8):c.2875C>T (p.Gln959Ter)-   NM_020920.3(CHD8):c.3172C>T (p.Arg1058Ter)-   NM_181332.2(NLGN4X):c.301C>T (p.Arg101Ter)-   NM_018094.4(GSPT2):c.1021G>A (p.Val341Ile)-   NM_000314.6(PTEN):c.392C>T (p.Thr131Ile)    See Table A. Accordingly, an aspect of the invention relates to a    method for treating or preventing Autism by correcting one or more    pathogenic G-to-A or C-to-T mutations/SNPs, particularly one or more    pathogenic G-to-A or C-to-T mutations/SNPs present in at least one    gene selected from MECP2, NLGN3, SLC9A9, EHMT1, CHD8, NLGN4X ,    GSPT2, and PTEN, and more particularly one or more pathogenic G-to-A    or C-to-T mutations/SNPs described above.

Amyotrophyic Lateral Sclerosis (ALS)

In some embodiments, the methods, systems, and compositions describedherein are used to correct one or more pathogenic G-to-A or C-to-Tmutations/SNPs associated with ALS. In some embodiments, the pathogenicmutations/SNPs are present in at least one gene selected from SOD1, VCP,UBQLN2, ERBB4, HNRNPA1, TUBA4A, SOD1, TARDBP, FIG4, OPTN, SETX, SPG11,FUS, VAPB, ANG, CHCHD10, SQSTM1, and TBK1, including at least thefollowings:

-   NM_000454.4(SOD1):c.289G>A (p.Asp97Asn)-   NM_007126.3(VCP):c.1774G>A (p.Asp592Asn)-   NM_007126.3(VCP):c.464G>A (p.Arg155His)-   NM_007126.3(VCP):c.572G>A (p.Arg191Gln)-   NM_013444.3(UBQLN2):c.1489C>T (p.Pro497Ser)-   NM_013444.3(UBQLN2):c.1525C>T (p.Pro509Ser)-   NM_013444.3(UBQLN2):c.1573C>T (p.Pro525Ser)-   NM_013444.3(UBQLN2):c.1490C>T (p.Pro497Leu)-   NM_005235.2(ERBB4):c.2780G>A (p.Arg927Gln)-   NM_005235.2(ERBB4):c.3823C>T (p.Arg1275Trp)-   NM_031157.3(HNRNPA1):c.940G>A (p.Asp314Asn)-   NM_006000.2(TUBA4A):c.643C>T (p.Arg215Cys)-   NM_006000.2(TUBA4A):c.958C>T (p.Arg320Cys)-   NM_006000.2(TUBA4A):c.959G>A (p.Arg320His)-   NM_006000.2(TUBA4A):c.1220G>A (p.Trp407Ter)-   NM_006000.2(TUBA4A):c.1147G>A (p.Ala383Thr)-   NM_000454.4(SOD1):c.112G>A (p.Gly38Arg)-   NM_000454.4(SOD1):c.124G>A (p.Gly42Ser)-   NM_000454.4(SOD1):c.125G>A (p.Gly42Asp)-   NM_000454.4(SOD1):c.14C>T (p.Ala5Val)-   NM_000454.4(SOD1):c.13G>A (p.Ala5Thr)-   NM_000454.4(SOD1):c.436G>A (p.Ala146Thr)-   NM_000454.4(SOD1):c.64G>A (p.Glu22Lys)-   NM_000454.4(SOD1):c.404G>A (p.Ser135Asn)-   NM_000454.4(SOD1):c.49G>A (p.Gly17Ser)-   NM_000454.4(SOD1):c.217G>A (p.Gly73Ser)-   NM_007375.3(TARDBP):c.892G>A (p.Gly298Ser)-   NM_007375.3(TARDBP):c.943G>A (p.Ala315Thr)-   NM_007375.3(TARDBP):c.883G>A (p.Gly295Ser)-   NM_007375.3(TARDBP):c.*697G>A-   NM_007375.3(TARDBP):c.1144G>A (p.Ala382Thr)-   NM_007375.3(TARDBP):c.859G>A (p.Gly287Ser)-   NM_014845.5(FIG4):c.547C>T (p.Arg183Ter)-   NM_001008211.1(OPTN):c.1192C>T (p.Gln398Ter)-   NM_015046.5(SETX):c.6407G>A (p.Arg2136His)-   NM_015046.5(SETX):c.8C>T (p.Thr3Ile)-   NM_025137.3(SPG11):c.118C>T (p.Gln40Ter)-   NM_025137.3(SPG11):c.267G>A (p.Trp89Ter)-   NM_025137.3(SPG11):c.5974C>T (p.Arg1992Ter)-   NM_004960.3(FUS):c.1553G>A (p.Arg518Lys)-   NM_004960.3(FUS):c.1561C>T (p.Arg521Cys)-   NM_004960.3(FUS):c.1562G>A (p.Arg521His)-   NM_004960.3(FUS):c.1520G>A (p.Gly507Asp)-   NM_004960.3(FUS):c.1483C>T (p.Arg495Ter)-   NM_004960.3(FUS):c.616G>A (p.Gly206Ser)-   NM_004960.3(FUS):c.646C>T (p.Arg216Cys)-   NM_004738.4(VAPB):c.166C>T (p.Pro56Ser)-   NM_004738.4(VAPB):c.137C>T (p.Thr46Ile)-   NM_001145.4(ANG):c.164G>A (p.Arg55Lys)-   NM_001145.4(ANG):c.155G>A (p.Ser52Asn)-   NM_001145.4(ANG):c.407C>T (p.Pro136Leu)-   NM_001145.4(ANG):c.409G>A (p.Val137Ile)-   NM_001301339.1(CHCHD10):c.239C>T (p.Pro80Leu)-   NM_001301339.1(CHCHD10):c.176C>T (p.Ser59Leu)-   NM_001142298.1(SQSTM1):c.-47-1924C>T-   NM_003900.4(SQSTM1):c.1160C>T (p.Pro387Leu)-   NM_003900.4(SQSTM1):c.1175C>T (p.Pro392Leu)-   NM_013254.3(TBK1):c.1340+1G>A-   NM_013254.3(TBK1):c.2086G>A (p.Glu696Lys)    See Table A. Accordingly, an aspect of the invention relates to a    method for treating or preventing ALS by correcting one or more    pathogenic G-to-A or C-to-T mutations/SNPs, particularly one or more    pathogenic G-to-A or C-to-T mutations/SNPs present in at least one    gene selected from SOD1, VCP, UBQLN2, ERBB4, HNRNPA1, TUBA4A, SOD1,    TARDBP, FIG4, OPTN, SETX, SPG11, FUS, VAPB, ANG, CHCHD10, SQSTM1,    and TBK1, and more particularly one or more pathogenic G-to-A or    C-to-T mutations/SNPs described above.

Schizophrenia

In some embodiments, the methods, systems, and compositions describedherein are used to correct one or more pathogenic G-to-A or C-to-Tmutations/SNPs associated with Schizophrenia. In some embodiment, thepathogenic mutations/SNPs are present in at least one gene selected fromPRODH, SETD1A, and SHANK3, including at least the followings:

-   NM_016335.4(PRODH):c.1292G>A (p.Arg431His)-   NM_016335.4(PRODH):c.1397C>T (p.Thr466Met)-   NM_014712.2(SETD1A):c.2209C>T (p.Gln737Ter)-   NM_033517.1(SHANK3):c.3349C>T (p.Arg1117Ter)-   NM_033517.1(SHANK3):c.1606C>T (p.Arg536Trp)    See Table A. Accordingly, an aspect of the invention relates to a    method for treating or preventing Schizophrenia by correcting one or    more pathogenic G-to-A or C-to-T mutations/SNPs, particularly one or    more pathogenic G-to-A or C-to-T mutations/SNPs present in at least    one gene selected from PRODH, SETD1A, and SHANK3, and more    particularly one or more pathogenic G-to-A or C-to-T mutations/SNPs    described above.

Adrenoleukodystrophy

In some embodiments, the methods, systems, and compositions describedherein are used to correct one or more pathogenic G-to-A or C-to-Tmutations/SNPs associated with Adrenoleukodystrophy. In some embodiment,the pathogenic mutations/SNPs are present in at least the ABCD1 gene,including at least the followings:

-   NM_000033.3(ABCD1):c.421G>A (p.Ala141Thr)-   NM_000033.3(ABCD1):c.796G>A (p.Gly266Arg)-   NM_000033.3(ABCD1):c.1252C>T (p.Arg418Trp)-   NM_000033.3(ABCD1):c.1552C>T (p.Arg518Trp)-   NM_000033.3(ABCD1):c.1850G>A (p.Arg617His)-   NM_000033.3(ABCD1):c.1396C>T (p.Gln466Ter)-   NM_000033.3(ABCD1):c.1553G>A (p.Arg518Gln)-   NM_000033.3(ABCD1):c.1679C>T (p.Pro560Leu)-   NM_000033.3(ABCD1):c.1771C>T (p.Arg591Trp)-   NM_000033.3(ABCD1):c.1802G>A (p.Trp601Ter)-   NM_000033.3(ABCD1):c.346G>A (p.Gly116Arg)-   NM_000033.3(ABCD1):c.406C>T (p.Gln136Ter)-   NM_000033.3(ABCD1):c.1661G>A (p.Arg554His)-   NM_000033.3(ABCD1):c.1825G>A (p.Glu609Lys)-   NM_000033.3(ABCD1):c.1288C>T (p.Gln430Ter)-   NM_000033.3(ABCD1):c.1781-1G>A-   NM_000033.3(ABCD1):c.529C>T (p.Gln177Ter)-   NM_000033.3(ABCD1):c.1866-10G>A    See Table A. Accordingly, an aspect of the invention relates to a    method for treating or preventing Adrenoleukodystrophy by correcting    one or more pathogenic G-to-A or C-to-T mutations/SNPs, particularly    one or more pathogenic G-to-A or C-to-T mutations/SNPs present in at    least the ABCD1 gene, and more particularly one or more pathogenic    G-to-A or C-to-T mutations/SNPs described above.

Aicardi Goutieres Syndrome

In some embodiments, the methods, systems, and compositions describedherein are used to correct one or more pathogenic G-to-A or C-to-Tmutations/SNPs associated with Aicardi Goutieres syndrome. In someembodiment, the pathogenic mutations/SNPs are present in at least onegene selected from TREX1, RNASEH2C, ADAR, and IFIH1, including at leastthe followings:

-   NM_016381.5(TREX1):c.794G>A (p.Trp265Ter)-   NM_033629.4(TREX1):c.52G>A (p.Asp18Asn)-   NM_033629.4(TREX1):c.490C>T (p.Arg164Ter)-   NM_032193.3(RNASEH2C):c.205C>T (p.Arg69Trp)-   NM_001111.4(ADAR):c.3019G>A (p.Gly1007Arg)-   NM_022168.3(IFIH1):c.2336G>A (p.Arg779His)-   NM_022168.3(IFIH1):c.2335C>T (p.Arg779Cys)    See Table A. Accordingly, an aspect of the invention relates to a    method for treating or preventing Aicardi Goutieres syndrome by    correcting one or more pathogenic G-to-A or C-to-T mutations/SNPs,    particularly one or more pathogenic G-to-A or C-to-T mutations/SNPs    present in at least one gene selected from TREX1, RNASEH2C, ADAR,    and IFIH1, and more particularly one or more pathogenic G-to-A or    C-to-T mutations/SNPs described above.

Fabry Disease

In some embodiments, the methods, systems, and compositions describedherein are used to correct one or more pathogenic G-to-A or C-to-Tmutations/SNPs associated with Fabry disease. In some embodiment, thepathogenic mutations/SNPs are present in at least the GLA gene,including at least the followings:

-   NM_000169.2(GLA):c.1024C>T (p.Arg342Ter)-   NM_000169.2(GLA):c.1066C>T (p.Arg356Trp)-   NM_000169.2(GLA):c.1025G>A (p.Arg342Gln)-   NM_000169.2(GLA):c.281G>A (p.Cys94Tyr)-   NM_000169.2(GLA):c.677G>A (p.Trp226Ter)-   NM_000169.2(GLA):c.734G>A (p.Trp245Ter)-   NM_000169.2(GLA):c.748C>T (p.Gln250Ter)-   NM_000169.2(GLA):c.658C>T (p.Arg220Ter)-   NM_000169.2(GLA):c.730G>A (p.Asp244Asn)-   NM_000169.2(GLA):c.369+1G>A-   NM_000169.2(GLA):c.335G>A (p.Arg112His)-   NM_000169.2(GLA):c.485G>A (p.Trp162Ter)-   NM_000169.2(GLA):c.661C>T (p.Gln221Ter)-   NM_000169.2(GLA):c.916C>T (p.Gln306Ter)-   NM_000169.2(GLA):c.1072G>A (p.Glu358Lys)-   NM_000169.2(GLA):c.1087C>T (p.Arg363Cys)-   NM_000169.2(GLA):c.1088G>A (p.Arg363His)-   NM_000169.2(GLA):c.605G>A (p.Cys202Tyr)-   NM_000169.2(GLA):c.830G>A (p.Trp277Ter)-   NM_000169.2(GLA):c.979C>T (p.Gln327Ter)-   NM_000169.2(GLA):c.422C>T (p.Thr141Ile)-   NM_000169.2(GLA):c.285G>A (p.Trp95Ter)-   NM_000169.2(GLA):c.735G>A (p.Trp245Ter)-   NM_000169.2(GLA):c.639+919G>A-   NM_000169.2(GLA):c.680G>A (p.Arg227Gln)-   NM_000169.2(GLA):c.679C>T (p.Arg227Ter)-   NM_000169.2(GLA):c.242G>A (p.Trp81Ter)-   NM_000169.2(GLA):c.901C>T (p.Arg301Ter)-   NM_000169.2(GLA):c.974G>A (p.Gly325Asp)-   NM_000169.2(GLA):c.847C>T (p.Gln283Ter)-   NM_000169.2(GLA):c.469C>T (p.Gln157Ter)-   NM_000169.2(GLA):c.1118G>A (p.Gly373Asp)    See Table A. Accordingly, an aspect of the invention relates to a    method for treating or preventing Fabry disease by correcting one or    more pathogenic G-to-A or C-to-T mutations/SNPs, particularly one or    more pathogenic G-to-A or C-to-T mutations/SNPs present in at least    the GLA gene, and more particularly one or more pathogenic G-to-A or    C-to-T mutations/SNPs described above.

Lesch-Nyhan Syndrome

In some embodiments, the methods, systems, and compositions describedherein are used to correct one or more pathogenic G-to-A or C-to-Tmutations/SNPs associated with Lesch-Nyhan syndrome. In some embodiment,the pathogenic mutations/SNPs are present in at least the HPRT1 gene,including at least the followings:

-   NM_000194.2(HPRT1):c.151C>T (p.Arg51Ter)-   NM_000194.2(HPRT1):c.384+1G>A    See Table A. Accordingly, an aspect of the invention relates to a    method for treating or preventing Lesch-Nyhan syndrome by correcting    one or more pathogenic G-to-A or C-to-T mutations/SNPs, particularly    one or more pathogenic G-to-A or C-to-T mutations/SNPs present in at    least the HPRT1 gene, and more particularly one or more pathogenic    G-to-A or C-to-T mutations/SNPs described above.

Menkes Disease

In some embodiments, the methods, systems, and compositions describedherein are used to correct one or more pathogenic G-to-A or C-to-Tmutations/SNPs associated with Menkes Disease. In some embodiment, thepathogenic mutations/SNPs are present in at least the ATP7A gene,including at least the followings:

-   NM_000052.6(ATP7A):c.601C>T (p.Arg201Ter)-   NM_000052.6(ATP7A):c.2938C>T (p.Arg980Ter)-   NM_000052.6(ATP7A):c.3056G>A (p.Gly1019Asp)-   NM_000052.6(ATP7A):c.598C>T (p.Gln200Ter)-   NM_000052.6(ATP7A):c.1225C>T (p.Arg409Ter)-   NM_000052.6(ATP7A):c.1544-1G>A-   NM_000052.6(ATP7A):c.1639C>T (p.Arg547Ter)-   NM_000052.6(ATP7A):c.1933C>T (p.Arg645Ter)-   NM_000052.6(ATP7A):c.1946+5G>A-   NM_000052.6(ATP7A):c.1950G>A (p.Trp650Ter)-   NM_000052.6(ATP7A):c.2179G>A (p.Gly727Arg)-   NM_000052.6(ATP7A):c.2187G>A (p.Trp729Ter)-   NM_000052.6(ATP7A):c.2383C>T (p.Arg795Ter)-   NM_000052.6(ATP7A):c.2499-1G>A-   NM_000052.6(ATP7A):c.2555C>T (p.Pro852Leu)-   NM_000052.6(ATP7A):c.2956C>T (p.Arg986Ter)-   NM_000052.6(ATP7A):c.3112-1G>A-   NM_000052.6(ATP7A):c.3466C>T (p.Gln1156Ter)-   NM_000052.6(ATP7A):c.3502C>T (p.Gln1168Ter)-   NM_000052.6(ATP7A):c.3764G>A (p.Gly1255Glu)-   NM_000052.6(ATP7A):c.3943G>A (p.Gly1315Arg)-   NM_000052.6(ATP7A):c.4123+1G>A-   NM_000052.6(ATP7A):c.4226+5G>A    See Table A. Accordingly, an aspect of the invention relates to a    method for treating or preventing Menkes Disease by correcting one    or more pathogenic G-to-A or C-to-T mutations/SNPs, particularly one    or more pathogenic G-to-A or C-to-T mutations/SNPs present in at    least the ATP7A gene, and more particularly one or more pathogenic    G-to-A or C-to-T mutations/SNPs described above.

Eye Diseases

Pathogenic G-to-A or C-to-T mutations/SNPs associated with various eyediseases are reported in the ClinVar database and disclosed in Table A,including but not limited to Stargardt Disease, Bardet-Biedl Syndrome,Cone-rod dystrophy, Congenital Stationary Night Blindness, UsherSyndrome, Leber Congenital Amaurosis, Retinitis Pigmentosa, andAchromatopsia. Accordingly, an aspect of the invention relates to amethod for correcting one or more pathogenic G-to-A or C-to-Tmutations/SNPs associated with any of these diseases, as discussedbelow.

Stargardt Disease

In some embodiments, the methods, systems, and compositions describedherein are used to correct one or more pathogenic G-to-A or C-to-Tmutations/SNPs associated with Stargardt Disease. In some embodiment,the pathogenic mutations/SNPs are present in the ABCA4 gene, includingat least the followings:

-   NM_000350.2(ABCA4):c.4429C>T (p.Gln1477Ter)-   NM_000350.2(ABCA4):c.6647C>T (p.Ala2216Val)-   NM_000350.2(ABCA4):c.5312+1G>A-   NM_000350.2(ABCA4):c.5189G>A (p.Trp1730Ter)-   NM_000350.2(ABCA4):c.4352+1G>A-   NM_000350.2(ABCA4):c.4253+5G>A-   NM_000350.2(ABCA4):c.3871C>T (p.Gln1291Ter)-   NM_000350.2(ABCA4):c.3813G>A (p.Glu1271=)-   NM_000350.2(ABCA4):c.1293G>A (p.Trp431Ter)-   NM_000350.2(ABCA4):c.206G>A (p.Trp69Ter)-   NM_000350.2(ABCA4):c.3322C>T (p.Arg1108Cys)-   NM_000350.2(ABCA4):c.1804C>T (p.Arg602Trp)-   NM_000350.2(ABCA4):c.1937+1G>A-   NM_000350.2(ABCA4):c.2564G>A (p.Trp855Ter)-   NM_000350.2(ABCA4):c.4234C>T (p.Gln1412Ter)-   NM_000350.2(ABCA4):c.4457C>T (p.Pro1486Leu)-   NM_000350.2(ABCA4):c.4594G>A (p.Asp1532Asn)-   NM_000350.2(ABCA4):c.4919G>A (p.Arg1640Gln)-   NM_000350.2(ABCA4):c.5196+1G>A-   NM_000350.2(ABCA4):c.6316C>T (p.Arg2106Cys)-   NM_000350.2(ABCA4):c.3056C>T (p.Thr1019Met)-   NM_000350.2(ABCA4):c.52C>T (p.Arg18Trp)-   NM_000350.2(ABCA4):c.122G>A (p.Trp41Ter)-   NM_000350.2(ABCA4):c.1903C>T (p.Gln635Ter)-   NM_000350.2(ABCA4):c.194G>A (p.Gly65Glu)-   NM_000350.2(ABCA4):c.3085C>T (p.Gln1029Ter)-   NM_000350.2(ABCA4):c.4195G>A (p.Glu1399Lys)-   NM_000350.2(ABCA4):c.454C>T (p.Arg152Ter)-   NM_000350.2(ABCA4):c.45G>A (p.Trp15Ter)-   NM_000350.2(ABCA4):c.4610C>T (p.Thr1537Met)-   NM_000350.2(ABCA4):c.6112C>T (p.Arg2038Trp)-   NM_000350.2(ABCA4):c.6118C>T (p.Arg2040Ter)-   NM_000350.2(ABCA4):c.6342G>A (p.Val2114=)-   NM_000350.2(ABCA4):c.6658C>T (p.Gln2220Ter)    See Table A. Accordingly, an aspect of the invention relates to a    method for treating or preventing Stargardt Disease by correcting    one or more pathogenic G-to-A or C-to-T mutations/SNPs, particularly    one or more pathogenic G-to-A or C-to-T mutations/SNPs present in    the ABCA4 gene, and more particularly one or more pathogenic G-to-A    or C-to-T mutations/SNPs described above.

Bardet-Biedl Syndrome

In some embodiments, the methods, systems, and compositions describedherein are used to correct one or more pathogenic G-to-A or C-to-Tmutations/SNPs associated with Bardet-Biedl Syndrome. In someembodiment, the pathogenic mutations/SNPs are present in at least onegene selected from BBS1, BBS2, BBS7, BBS9, BBS10, BBS12, LZTFL1, andTRIM32, including at least the followings:

-   NM_024649.4(BBS1):c.416G>A (p.Trp139Ter)-   NM_024649.4(BBS1):c.871C>T (p.Gln291Ter)-   NM_198428.2(BBS9):c.263+1G>A-   NM_001178007.1(BBS12):c.1704G>A (p.Trp568Ter)-   NM_001276378.1(LZTFL1):c.271C>T (p.Arg91Ter)-   NM_031885.3(BBS2):c.1864C>T (p.Arg622Ter)-   NM_198428.2(BBS9):c.1759C>T (p.Arg587Ter)-   NM_198428.2(BBS9):c.1789+1G>A-   NM_024649.4(BB S1):c.432+1G>A-   NM_176824.2(BBS7):c.632C>T (p.Thr211Ile)-   NM_012210.3(TRIM32):c.388C>T (p.Pro130Ser)-   NM_031885.3(BBS2):c.823C>T (p.Arg275Ter)-   NM_024685.3(BBS10):c.145C>T (p.Arg49Trp)    See Table A. Accordingly, an aspect of the invention relates to a    method for treating or preventing Bardet-Biedl Syndrome by    correcting one or more pathogenic G-to-A or C-to-T mutations/SNPs,    particularly one or more pathogenic G-to-A or C-to-T mutations/SNPs    present in at least one gene selected from BBS1, BBS2, BBS7, BBS9,    BBS10, BBS12, LZTFL1, and TRIM32, and more particularly one or more    pathogenic G-to-A or C-to-T mutations/SNPs described above.

Cone-Rod Dystrophy

In some embodiments, the methods, systems, and compositions describedherein are used to correct one or more pathogenic G-to-A or C-to-Tmutations/SNPs associated with Cone-rod dystrophy. In some embodiment,the pathogenic mutations/SNPs are present in at least one gene selectedfrom RPGRIP1, DRAM2, ABCA4, ADAM9, and CACNA1F, including at least thefollowings:

-   NM_020366.3(RPGRIP1):c.154C>T (p.Arg52Ter)-   NM_178454.5(DRAM2):c.494G>A (p.Trp165Ter)-   NM_178454.5(DRAM2):c.131G>A (p.Ser44Asn)-   NM_000350.2(ABCA4):c.161G>A (p.Cys54Tyr)-   NM_000350.2(ABCA4):c.5714+5G>A-   NM_000350.2(ABCA4):c.880C>T (p.Gln294Ter)-   NM_000350.2(ABCA4):c.6079C>T (p.Leu2027Phe)-   NM_000350.2(ABCA4):c.3113C>T (p.Ala1038Val)-   NM_000350.2(ABCA4):c.634C>T (p.Arg212Cys)-   NM_003816.2(ADAM9):c.490C>T (p.Arg164Ter)-   NM_005183.3(CACNA1F):c.244C>T (p.Arg82Ter)    See Table A. Accordingly, an aspect of the invention relates to a    method for treating or preventing Cone-rod dystrophy by correcting    one or more pathogenic G-to-A or C-to-T mutations/SNPs, particularly    one or more pathogenic G-to-A or C-to-T mutations/SNPs present in at    least one gene selected from RPGRIP1, DRAM2, ABCA4, ADAM9, and    CACNA1F, and more particularly one or more pathogenic G-to-A or    C-to-T mutations/SNPs described above.

Congenital Stationary Night Blindness

In some embodiments, the methods, systems, and compositions describedherein are used to correct one or more pathogenic G-to-A or C-to-Tmutations/SNPs associated with Congenital Stationary Night Blindness. Insome embodiment, the pathogenic mutations/SNPs are present in at leastone gene selected from GRM6, TRPM1, GPR179, and CACNA1F, including atleast the followings:

-   NM_000843.3(GRM6):c.1462C>T (p.Gln488Ter)-   NM_002420.5(TRPM1):c.2998C>T (p.Arg1000Ter)-   NM_001004334.3(GPR179):c.673C>T (p.Gln225Ter)-   NM_005183.3(CACNA1F):c.2576+1G>A    See Table A. Accordingly, an aspect of the invention relates to a    method for treating or preventing Congenital Stationary Night    Blindness by correcting one or more pathogenic G-to-A or C-to-T    mutations/SNPs, particularly one or more pathogenic G-to-A or C-to-T    mutations/SNPs present in at least one gene selected from GRM6,    TRPM1, GPR179, and CACNA1F, and more particularly one or more    pathogenic G-to-A or C-to-T mutations/SNPs described above.

Usher Syndrome

In some embodiments, the methods, systems, and compositions describedherein are used to correct one or more pathogenic G-to-A or C-to-Tmutations/SNPs associated with Usher Syndrome. In some embodiment, thepathogenic mutations/SNPs are present in at least one gene selected fromMYO7A, USH1C, CDH23, PCDH15, USH2A, ADGRV1, WHRN, and CLRN1, includingat least the followings:

-   NM_000260.3(MYO7A):c.640G>A (p.Gly214Arg)-   NM_000260.3(MYO7A):c.1200+1G>A-   NM_000260.3(MYO7A):c.141G>A (p. Trp47Ter)-   NM_000260.3(MYO7A):c.1556G>A (p.Gly519Asp)-   NM_000260.3(MYO7A):c.1900C>T (p.Arg634Ter)-   NM_000260.3(MYO7A):c.1963C>T (p.Gln655Ter)-   NM_000260.3(MYO7A):c.2094+1G>A-   NM_000260.3(MYO7A):c.4293G>A (p.Trp1431Ter)-   NM_000260.3(MYO7A):c.5101C>T (p.Arg1701Ter)-   NM_000260.3(MYO7A):c.5617C>T (p.Arg1873Trp)-   NM_000260.3(MYO7A):c.5660C>T (p.Pro1887Leu)-   NM_000260.3(MYO7A):c.6070C>T (p.Arg2024Ter)-   NM_000260.3(MYO7A):c.470+1G>A-   NM_000260.3(MYO7A):c.5968C>T (p.Gln1990Ter)-   NM_000260.3(MYO7A):c.3719G>A (p.Arg1240Gln)-   NM_000260.3(MYO7A):c.494C>T (p.Thr165Met)-   NM_000260.3(MYO7A):c.5392C>T (p.Gln1798Ter)-   NM_000260.3(MYO7A):c.5648G>A (p.Arg1883Gln)-   NM_000260.3(MYO7A):c.448C>T (p.Arg150Ter)-   NM_000260.3(MYO7A):c.700C>T (p.Gln234Ter)-   NM_000260.3(MYO7A):c.635G>A (p.Arg212His)-   NM_000260.3(MYO7A):c.1996C>T (p.Arg666Ter)-   NM_005709.3(USH1C):c.216G>A (p.Val72=)-   NM_022124.5(CDH23):c.7362+5G>A-   NM_022124.5(CDH23):c.3481C>T (p.Arg1161Ter)-   NM_022124.5(CDH23):c.3628C>T (p.Gln1210Ter)-   NM_022124.5(CDH23):c.5272C>T (p.Gln1758Ter)-   NM_022124.5(CDH23):c.5712+1G>A-   NM_022124.5(CDH23):c.5712G>A (p.Thr1904=)-   NM_022124.5(CDH23):c.5923+1G>A-   NM_022124.5(CDH23):c.6049+1G>A-   NM_022124.5(CDH23):c.7776G>A (p.Trp2592Ter)-   NM_022124.5(CDH23):c.9556C>T (p.Arg3186Ter)-   NM_022124.5(CDH23):c.3706C>T (p.Arg1236Ter)-   NM_022124.5(CDH23):c.4309C>T (p.Arg1437Ter)-   NM_022124.5(CDH23):c.6050-9G>A-   NM_033056.3(PCDH15):c.3316C>T (p.Arg1106Ter)-   NM_033056.3(PCDH15):c.7C>T (p.Arg3Ter)-   NM_033056.3(PCDH15):c.1927C>T (p.Arg643Ter)-   NM_001142772.1(PCDH15):c.400C>T (p.Arg134Ter)-   NM_033056.3(PCDH15):c.3358C>T (p.Arg1120Ter)-   NM_206933.2(USH2A):c.11048-1G>A-   NM_206933.2(USH2A):c.1143+1G>A-   NM_206933.2(USH2A):c.11954G>A (p.Trp3985Ter)-   NM_206933.2(USH2A):c.12868C>T (p.Gln4290Ter)-   NM_206933.2(USH2A):c.14180G>A (p.Trp4727Ter)-   NM_206933.2(USH2A):c.14911C>T (p.Arg4971Ter)-   NM_206933.2(USH2A):c.5788C>T (p.Arg1930Ter)-   NM_206933.2(USH2A):c.5858-1G>A-   NM_206933.2(USH2A):c.6224G>A (p.Trp2075Ter)-   NM_206933.2(USH2A):c.820C>T (p.Arg274Ter)-   NM_206933.2(USH2A):c.8981G>A (p.Trp2994Ter)-   NM_206933.2(USH2A):c.9304C>T (p.Gln3102Ter)-   NM_206933.2(USH2A):c.13010C>T (p.Thr4337Met)-   NM_206933.2(USH2A):c.14248C>T (p.Gln4750Ter)-   NM_206933.2(USH2A):c.6398G>A (p.Trp2133Ter)-   NM_206933.2(USH2A):c.632G>A (p.Trp211Ter)-   NM_206933.2(USH2A):c.6601C>T (p.Gln2201Ter)-   NM_206933.2(USH2A):c.13316C>T (p.Thr4439Ile)-   NM_206933.2(USH2A):c.4405C>T (p.Gln1469Ter)-   NM_206933.2(USH2A):c.9570+1G>A-   NM_206933.2(USH2A):c.8740C>T (p.Arg2914Ter)-   NM_206933 .2(USH2A):c.8681+1G>A-   NM_206933.2(USH2A):c.1000C>T (p.Arg334Trp)-   NM_206933.2(USH2A):c.14175G>A (p.Trp4725Ter)-   NM_206933.2(USH2A):c.9390G>A (p.Trp3130Ter)-   NM_206933.2(USH2A):c.908G>A (p.Arg303His)-   NM_206933.2(USH2A):c.5776+1G>A-   NM_206933.2(USH2A):c.11156G>A (p.Arg3719His)-   NM_032119.3(ADGRV1):c.2398C>T (p.Arg800Ter)-   NM_032119.3(ADGRV1):c.7406G>A (p.Trp2469Ter)-   NM_032119.3(ADGRV1):c.12631C>T (p.Arg4211Ter)-   NM_032119.3(ADGRV1):c.7129C>T (p.Arg2377Ter)-   NM_032119.3(ADGRV1):c.14885G>A (p.Trp4962Ter)-   NM_015404.3(WHRN):c.1267C>T (p.Arg423Ter)-   NM_174878.2(CLRN1):c.619C>T (p.Arg207Ter)    See Table A. Accordingly, an aspect of the invention relates to a    method for treating or preventing Enhanced Usher Syndrome by    correcting one or more pathogenic G-to-A or C-to-T mutations/SNPs,    particularly one or more pathogenic G-to-A or C-to-T mutations/SNPs    present in at least one gene selected from MYO7A, USH1C, CDH23,    PCDH15, USH2A, ADGRV1, WHRN, and CLRN1, and more particularly one or    more pathogenic G-to-A or C-to-T mutations/SNPs described above.

Leber Congenital Amaurosis

In some embodiments, the methods, systems, and compositions describedherein are used to correct one or more pathogenic G-to-A or C-to-Tmutations/SNPs associated with Leber Congenital Amaurosis. In someembodiment, the pathogenic mutations/SNPs are present in at least onegene selected from TULP1, RPE65, SPATA7, AIPL1, CRB1, NMNAT1, and PEX1,including at least the followings:

-   NM_003322.5(TULP1):c.1495+1G>A-   NM_000329.2(RPE65):c.11+5G>A-   NM_018418.4(SPATA7):c.322C>T (p.Arg108Ter)-   NM_014336.4(AIPL1):c.784G>A (p.Gly262Ser)-   NM_201253.2(CRB1):c.1576C>T (p.Arg526Ter)-   NM_201253.2(CRB1):c.3307G>A (p.Gly1103Arg)-   NM_201253.2(CRB1):c.2843G>A (p.Cys948Tyr)-   NM_022787.3(NMNAT1):c.769G>A (p.Glu257Lys)-   NM_000466.2(PEX1):c.2528G>A (p.Gly843Asp)    See Table A. Accordingly, an aspect of the invention relates to a    method for treating or preventing Leber Congenital Amaurosis by    correcting one or more pathogenic G-to-A or C-to-T mutations/SNPs,    particularly one or more pathogenic G-to-A or C-to-T mutations/SNPs    present in at least one gene selected from TULP1, RPE65, SPATA7,    AIPL1, CRB1, NMNAT1, and PEX1, and more particularly one or more    pathogenic G-to-A or C-to-T mutations/SNPs described above.

Retinitis Pigmentosa

In some embodiments, the methods, systems, and compositions describedherein are used to correct one or more pathogenic G-to-A or C-to-Tmutations/SNPs associated with Retinitis Pigmentosa. In some embodiment,the pathogenic mutations/SNPs are present in at least one gene selectedfrom CRB1, IFT140, RP1, IMPDH1, PRPF31, RPGR, ABCA4, RPE65, EYS, NRL,FAM161A, NR2E3, USH2A, RHO, PDE6B, KLHL7, PDE6A, CNGB1, BEST1, C2orf71,PRPH2, CA4, CERKL, RPE65, PDE6B, and ADGRV1, including at least thefollowings:

-   NM_001257965.1(CRB1):c.2711G>A (p.Cys904Tyr)-   NM_014714.3(IFT140):c.3827G>A (p.Gly1276Glu)-   NM_006269.1(RP1):c.2029C>T (p.Arg677Ter)-   NM_000883.3(IMPDH1):c.931G>A (p.Asp311Asn)-   NM_015629.3(PRPF31):c.1273C>T (p.Gln425Ter)-   NM_015629.3(PRPF31):c.1073+1G>A-   NM_000328.2(RPGR):c.1387C>T (p.Gln463Ter)-   NM_000350.2(ABCA4):c.4577C>T (p.Thr1526Met)-   NM_000350.2(ABCA4):c.6229C>T (p.Arg2077Trp)-   NM_000329.2(RPE65):c.271C>T (p.Arg91Trp)-   NM_001142800.1(EYS):c.2194C>T (p.Gln732Ter)-   NM_001142800.1(EYS):c.490C>T (p.Arg164Ter)-   NM_006177.3(NRL):c.151C>T (p.Pro51Ser)-   NM_001201543.1(FAM161A):c.1567C>T (p.Arg523Ter)-   NM_014249.3(NR2E3):c.166G>A (p.Gly56Arg)-   NM_206933.2(USH2A):c.2209C>T (p.Arg737Ter)-   NM_206933.2(USH2A):c.14803C>T (p.Arg4935Ter)-   NM_206933.2(USH2A):c.10073G>A (p.Cys3358Tyr)-   NM_000539.3(RHO):c.541G>A (p.Glu181Lys)-   NM_000283.3(PDE6B):c.892C>T (p.Gln298Ter)-   NM_001031710.2(KLHL7):c.458C>T (p.Ala153Val)-   NM_000440.2(PDE6A):c.1926+1G>A-   NM_001297.4(CNGB1):c.2128C>T (p.Gln710Ter)-   NM_001297.4(CNGB 1):c.952C>T (p.Gln318Ter)-   NM_004183.3(BEST1):c.682G>A (p.Asp228Asn)-   NM_001029883.2(C2orf71):c.1828C>T (p.Gln610Ter)-   NM_000322.4(PRPH2):c.647C>T (p.Pro216Leu)-   NM_000717.4(CA4):c.40C>T (p.Arg14Trp)-   NM_201548.4(CERKL):c.769C>T (p.Arg257Ter)-   NM_000329.2(RPE65):c.118G>A (p.Gly40Ser)-   NM_000322.4(PRPH2):c.499G>A (p.Gly167Ser)-   NM_000539.3(RHO):c.403C>T (p.Arg135Trp)-   NM_000283.3(PDE6B):c.2193+1G>A-   NM_032119.3(ADGRV1):c.6901C>T (p.Gln2301Ter)    See Table A. Accordingly, an aspect of the invention relates to a    method for treating or preventing Retinitis Pigmentosa by correcting    one or more pathogenic G-to-A or C-to-T mutations/SNPs, particularly    one or more pathogenic G-to-A or C-to-T mutations/SNPs present in at    least one gene selected from CRB1, IFT140, RP1, IMPDH1, PRPF31,    RPGR, ABCA4, RPE65, EYS, NRL, FAM161A, NR2E3, USH2A, RHO, PDE6B,    KLHL7, PDE6A, CNGB1, BEST1, C2orf71, PRPH2, CA4, CERKL, RPE65,    PDE6B, and ADGRV1, and more particularly one or more pathogenic    G-to-A or C-to-T mutations/SNPs described above.

Achromatopsia

In some embodiments, the methods, systems, and compositions describedherein are used to correct one or more pathogenic G-to-A or C-to-Tmutations/SNPs associated with Achromatopsia. In some embodiment, thepathogenic mutations/SNPs are present in at least one gene selected fromCNGA3, CNGB3, and ATF6, including at least the followings:

-   NM_001298.2(CNGA3):c.847C>T (p.Arg283Trp)-   NM_001298.2(CNGA3):c.101+1G>A-   NM_001298.2(CNGA3):c.1585G>A (p.Val529Met)-   NM_019098.4(CNGB3):c.1578+1G>A-   NM_019098.4(CNGB3):c.607C>T (p.Arg203Ter)-   NM_019098.4(CNGB3):c.1119G>A (p.Trp373Ter)-   NM_007348.3(ATF6):c.970C>T (p.Arg324Cys)    See Table A. Accordingly, an aspect of the invention relates to a    method for treating or preventing Achromatopsia by correcting one or    more pathogenic G-to-A or C-to-T mutations/SNPs, particularly one or    more pathogenic G-to-A or C-to-T mutations/SNPs present in at least    one gene selected from CNGA3, CNGB3, and ATF6, and more particularly    one or more pathogenic G-to-A or C-to-T mutations/SNPs described    above.

Diseases Affecting Hearing

Pathogenic G-to-A or C-to-T mutations/SNPs associated with variousdiseases affecting hearing are reported in the ClinVar database anddisclosed in Table A, including but not limited to deafness andNonsyndromic hearing loss. Accordingly, an aspect of the inventionrelates to a method for correcting one or more pathogenic G-to-A orC-to-T mutations/SNPs associated with any of these diseases, asdiscussed below.

Deafness

In some embodiments, the methods, systems, and compositions describedherein are used to correct one or more pathogenic G-to-A or C-to-Tmutations/SNPs associated with deafness. In some embodiment, thepathogenic mutations/SNPs are present in at least one gene selected fromFGF3, MYO7A, STRC, ACTG1, SLC17A8, TMC1, GJB2, MYH14, COCH, CDH23,USH1C, GJB2, MYO7A, PCDH15, MYO15A, MYO3A, WHRN, DFNB59, TMC1, LOXHD1,TMPRSS3, OTOGL, OTOF, JAG1, and MARVELD2, including at least thefollowings:

-   NM_005247.2(FGF3):c.283C>T (p.Arg95Trp)-   NM_000260.3(MYO7A):c.652G>A (p.Asp218Asn)-   NM_000260.3(MYO7A):c.689C>T (p.Ala230Val)-   NM_153700.2(STRC):c.4057C>T (p.Gln1353Ter)-   NM_001614.3(ACTG1):c.721G>A (p.Glu241Lys)-   NM_139319.2(SLC17A8):c.632C>T (p.Ala211Val)-   NM_138691.2(TMC1):c.1714G>A (p.Asp572Asn)-   NM_004004.5(GJB2):c.598G>A (p.Gly200Arg)-   NM_004004.5(GJB2):c.71G>A (p.Trp24Ter)-   NM_004004.5(GJB2):c.416G>A (p.Ser139Asn)-   NM_004004.5(GJB2):c.224G>A (p.Arg75Gln)-   NM_004004.5(GJB2):c.95G>A (p.Arg32His)-   NM_004004.5(GJB2):c.250G>A (p.Val84Met)-   NM_004004.5(GJB2):c.428G>A (p.Arg143Gln)-   NM_004004.5(GJB2):c.551G>A (p.Arg184Gln)-   NM_004004.5(GJB2):c.223C>T (p.Arg75Trp)-   NM_024729.3(MYH14):c.359C>T (p.Ser120Leu)-   NM_004086.2(COCH):c.151C>T (p.Pro51Ser)-   NM_022124.5(CDH23):c.4021G>A (p.Asp1341Asn)-   NM_153700.2(STRC):c.4701+1G>A-   NM_153676.3(USH1C):c.496+1G>A-   NM_004004.5(GJB2):c.131G>A (p.Trp44Ter)-   NM_004004.5(GJB2):c.283G>A (p.Val95Met)-   NM_004004.5(GJB2):c.298C>T (p.His100Tyr)-   NM_004004.5(GJB2):c.427C>T (p.Arg143Trp)-   NM_004004.5(GJB2):c.109G>A (p.Val37Ile)-   NM_004004.5(GJB2):c.-23+1G>A-   NM_004004.5(GJB2):c.148G>A (p.Asp50Asn)-   NM_004004.5(GJB2):c.134G>A (p.Gly45Glu)-   NM_004004.5(GJB2):c.370C>T (p.Gln124Ter)-   NM_004004.5(GJB2):c.230G>A (p.Trp77Ter)-   NM_004004.5(GJB2):c.231G>A (p.Trp77Ter)-   NM_000260.3(MYO7A):c.5899C>T (p.Arg1967Ter)-   NM_000260.3(MYO7A):c.2005C>T (p.Arg669Ter)-   NM_033056.3(PCDH15):c.733C>T (p.Arg245Ter)-   NM_016239.3(MYO15A):c.3866+1G>A-   NM_016239.3(MYO15A):c.6178-1G>A-   NM_016239.3(MYO15A):c.8714-1G>A-   NM_017433.4(MYO3A):c.2506-1G>A-   NM_015404.3(WHRN):c.1417-1G>A-   NM_001042702.3(DFNB59):c.499C>T (p.Arg167Ter)-   NM_138691.2(TMC1):c.100C>T (p.Arg34Ter)-   NM_138691.2(TMC1):c.1165C>T (p.Arg389Ter)-   NM_144612.6(LOXHD1):c.2008C>T (p.Arg670Ter)-   NM_144612.6(LOXHD1):c.4714C>T (p.Arg1572Ter)-   NM_144612.6(LOXHD1):c.4480C>T (p.Arg1494Ter)-   NM_024022.2(TMPRSS3):c.325C>T (p.Arg109Trp)-   NM_173591.3(OTOGL):c.3076C>T (p.Gln1026Ter)-   NM_194248.2(OTOF):c.4483C>T (p.Arg1495Ter)-   NM_194248.2(OTOF):c.2122C>T (p.Arg708Ter)-   NM_194248.2(OTOF):c.2485C>T (p.Gln829Ter)-   NM_001038603.2(MARVELD2):c.1498C>T (p.Arg500Ter)    See Table A. Accordingly, an aspect of the invention relates to a    method for treating or preventing deafness by correcting one or more    pathogenic G-to-A or C-to-T mutations/SNPs, particularly one or more    pathogenic G-to-A or C-to-T mutations/SNPs present in at least one    gene selected from FGF3, MYO7A, STRC, ACTG1, SLC17A8, TMC1, GJB2,    MYH14, COCH, CDH23, USH1C, GJB2, MYO7A, PCDH15, MYO15A, MYO3A, WHRN,    DFNB59, TMC1, LOXHD1, TMPRSS3, OTOGL, OTOF, JAG1, and MARVELD2, and    more particularly one or more pathogenic G-to-A or C-to-T    mutations/SNPs described above.

Nonsyndromic Hearing Loss

In some embodiments, the methods, systems, and compositions describedherein are used to correct one or more pathogenic G-to-A or C-to-Tmutations/SNPs associated with Nonsyndromic hearing loss. In someembodiment, the pathogenic mutations/SNPs are present in at least onegene selected from GJB2, POU3F4, MYO15A, TMPRSS3, LOXHD1, OTOF, MYO6,OTOA, STRC, TRIOBP, MARVELD2, TMC1, TECTA, OTOGL, and GIPC3, includingat least the followings:

-   NM_004004.5(GJB2):c.169C>T (p.Gln57Ter)-   NM_000307.4(POU3F4):c.499C>T (p.Arg167Ter)-   NM_016239.3(MYO15A):c.8767C>T (p.Arg2923Ter)-   NM_024022.2(TMPRSS3):c.323-6G>A-   NM_024022.2(TMPRSS3):c.916G>A (p.Ala306Thr)-   NM_144612.6(LOXHD1):c.2497C>T (p.Arg833Ter)-   NM_194248.2(OTOF):c.2153G>A (p.Trp718Ter)-   NM_194248.2(OTOF):c.2818C>T (p.Gln940Ter)-   NM_194248.2(OTOF):c.4799+1G>A-   NM_004999.3(MYO6):c.826C>T (p.Arg276Ter)-   NM_144672.3(OTOA):c.1880+1G>A-   NM_153700.2(STRC):c.5188C>T (p.Arg1730Ter)-   NM_153700.2(STRC):c.3670C>T (p.Arg1224Ter)-   NM_153700.2(STRC):c.4402C>T (p.Arg1468Ter)-   NM_024022.2(TMPRSS3):c.1192C>T (p.Gln398Ter)-   NM_001039141.2(TRIOBP):c.6598C>T (p.Arg2200Ter)-   NM_016239.3(MYO15A):c.7893+1G>A-   NM_016239.3(MYO15A):c.5531+1G>A-   NM_016239.3(MYO15A):c.6046+1G>A-   NM_144612.6(LOXHD1):c.3169C>T (p.Arg1057Ter)-   NM_001038603 .2(MARVELD2):c.1331+1G>A-   NM_138691.2(TMC1):c.1676G>A (p.Trp559Ter)-   NM_138691.2(TMC1):c.1677G>A (p.Trp559Ter)-   NM_005422.2(TECTA):c.5977C>T (p.Arg1993Ter)-   NM_173591.3(OTOGL):c.4987C>T (p.Arg1663Ter)-   NM_153700.2(STRC):c.3493C>T (p.Gln1165Ter)-   NM_153700.2(STRC):c.3217C>T (p.Arg1073Ter)-   NM_016239.3(MYO15A):c.5896C>T (p.Arg1966Ter)-   NM_133261.2(GIPC3):c.411+1G>A    See Table A. Accordingly, an aspect of the invention relates to a    method for treating or preventing Nonsyndromic hearing loss by    correcting one or more pathogenic G-to-A or C-to-T mutations/SNPs,    particularly one or more pathogenic G-to-A or C-to-T mutations/SNPs    present in at least one gene selected from GJB2, POU3F4, MYO15A,    TMPRSS3, LOXHD1, OTOF, MYO6, OTOA, STRC, TRIOBP, MARVELD2, TMC1,    TECTA, OTOGL, and GIPC3, and more particularly one or more    pathogenic G-to-A or C-to-T mutations/SNPs described above.

Blood Disorders

Pathogenic G-to-A or C-to-T mutations/SNPs associated with various blooddisorders are reported in the ClinVar database and disclosed in Table A,including but not limited to Beta-thalassemia, Hemophilia A, HemophiliaB, Hemophilia C, and Wiskott-Aldrich syndrome. Accordingly, an aspect ofthe invention relates to a method for correcting one or more pathogenicG-to-A or C-to-T mutations/SNPs associated with any of these diseases,as discussed below.

Beta-Thalassemia

In some embodiments, the methods, systems, and compositions describedherein are used to correct one or more pathogenic G-to-A or C-to-Tmutations/SNPs associated with Beta-thalassemia. In some embodiment, thepathogenic mutations/SNPs are present in at least the HBB gene,including at least the followings:

-   NM_000518.4(HBB):c.-137C>T-   NM_000518.4(HBB):c.-50-88C>T-   NM_000518.4(HBB):c.-140C>T-   NM_000518.4(HBB):c.316-197C>T-   NM_000518.4(HBB):c.93-21G>A-   NM_000518.4(HBB):c.114G>A (p.Trp38Ter)-   NM_000518.4(HBB):c.118C>T (p.Gln40Ter)-   NM_000518.4(HBB):c.92+1G>A-   NM_000518.4(HBB):c.315+1G>A-   NM_000518.4(HBB):c.92+5G>A-   NM_000518.4(HBB):c.-50-101C>T    See Table A. Accordingly, an aspect of the invention relates to a    method for treating or preventing Beta-thalassemia by correcting one    or more pathogenic G-to-A or C-to-T mutations/SNPs, particularly one    or more pathogenic G-to-A or C-to-T mutations/SNPs present in the    HBB gene, and more particularly one or more pathogenic G-to-A or    C-to-T mutations/SNPs described above.

Hemophilia A

In some embodiments, the methods, systems, and compositions describedherein are used to correct one or more pathogenic G-to-A or C-to-Tmutations/SNPs associated with Hemophilia A. In some embodiment, thepathogenic mutations/SNPs are present in at least the F8 gene, includingat least the followings:

-   NM_000132.3(F8):c.3169G>A (p.Glu1057Lys)-   NM_000132.3(F8):c.902G>A (p.Arg301His)-   NM_000132.3(F8):c.1834C>T (p.Arg612Cys)    See Table A. Accordingly, an aspect of the invention relates to a    method for treating or preventing Hemophilia A by correcting one or    more pathogenic G-to-A or C-to-T mutations/SNPs, particularly one or    more pathogenic G-to-A or C-to-T mutations/SNPs present in the F8    gene, and more particularly one or more pathogenic G-to-A or C-to-T    mutations/SNPs described above.

Factor V Leiden

In some embodiments, the methods, systems, and compositions describedherein are used to correct Factor V Leiden mutations. Thisdisease-causing single point mutation (G1746→A) represents the mostabundant genetic risk factor in heritable multifactorial thrombophiliain the Caucasian population. Due to the point mutation, a single aminoacid substitution (R534[RIGHTWARDS ARROW]Q) appears at the Protein Cdependent proteolytic cleavage site (R533R534) of the blood coagulationfactor F5. Whereas the heterozygous defect is accompanied by an onlyminor increase in thrombosis risk (ca. 8-fold), the homozygous defecthas a much more pronounced effect (>80-fold increased risk).19 DirectedRNA editing has the potential to compensate for this genetic defect byits repair at the RNA level.

Hemophilia B

In some embodiments, the methods, systems, and compositions describedherein are used to correct one or more pathogenic G-to-A or C-to-Tmutations/SNPs associated with Hemophilia B. In some embodiment, thepathogenic mutations/SNPs are present in at least the F9 gene, includingat least the followings:

-   NM_000133.3(F9):c.835G>A (p.Ala279Thr)    See Table A. Accordingly, an aspect of the invention relates to a    method for treating or preventing Hemophilia B by correcting one or    more pathogenic G-to-A or C-to-T mutations/SNPs, particularly one or    more pathogenic G-to-A or C-to-T mutations/SNPs present in the F9    gene, and more particularly one or more pathogenic G-to-A or C-to-T    mutations/SNPs described above.

Hemophilia C

In some embodiments, the methods, systems, and compositions describedherein are used to correct one or more pathogenic G-to-A or C-to-Tmutations/SNPs associated with Hemophilia C. In some embodiment, thepathogenic mutations/SNPs are present in at least the F11 gene,including at least the followings:

-   NM_000128.3(F11):c.400C>T (p.Gln134Ter)-   NM_000128.3(F11):c.1432G>A (p.Gly478Arg)-   NM_000128.3(F11):c.1288G>A (p.Ala430Thr)-   NM_000128.3(F11):c.326-1G>A    See Table A. Accordingly, an aspect of the invention relates to a    method for treating or preventing Hemophilia C by correcting one or    more pathogenic G-to-A or C-to-T mutations/SNPs, particularly one or    more pathogenic G-to-A or C-to-T mutations/SNPs present in the F 11    gene, and more particularly one or more pathogenic G-to-A or C-to-T    mutations/SNPs described above.

Wiskott-Aldrich Syndrome

In some embodiments, the methods, systems, and compositions describedherein are used to correct one or more pathogenic G-to-A or C-to-Tmutations/SNPs associated with Wiskott-Aldrich syndrome. In someembodiment, the pathogenic mutations/SNPs are present in at least theWAS gene, including at least the followings:

-   NM_000377.2(WAS):c.37C>T (p.Arg13Ter)-   NM_000377.2(WAS):c.257G>A (p.Arg86His)-   NM_000377.2(WAS):c.777+1G>A    See Table A. Accordingly, an aspect of the invention relates to a    method for treating or preventing Wiskott-Aldrich syndrome by    correcting one or more pathogenic G-to-A or C-to-T mutations/SNPs,    particularly one or more pathogenic G-to-A or C-to-T mutations/SNPs    present in the WAS gene, and more particularly one or more    pathogenic G-to-A or C-to-T mutations/SNPs described above.

Liver Diseases

Pathogenic G-to-A or C-to-T mutations/SNPs associated with various liverdiseases are reported in the ClinVar database and disclosed in Table A,including but not limited to Transthyretin amyloidosis,Alpha-1-antitrypsin deficiency, Wilson's disease, and Phenylketonuria.Accordingly, an aspect of the invention relates to a method forcorrecting one or more pathogenic G-to-A or C-to-T mutations/SNPsassociated with any of these diseases, as discussed below.

Transthyretin Amyloidosis

In some embodiments, the methods, systems, and compositions describedherein are used to correct one or more pathogenic G-to-A or C-to-Tmutations/SNPs associated with Transthyretin amyloidosis. In someembodiment, the pathogenic mutations/SNPs are present in at least theTTR gene, including at least the followings:

-   NM_000371.3(TTR):c.424G>A (p.Val142Ile)-   NM_000371.3(TTR):c.148G>A (p.Val50Met)-   NM_000371.3(TTR):c.118G>A (p.Val40Ile)    See Table A. Accordingly, an aspect of the invention relates to a    method for treating or preventing Transthyretin amyloidosis by    correcting one or more pathogenic G-to-A or C-to-T mutations/SNPs,    particularly one or more pathogenic G-to-A or C-to-T mutations/SNPs    present in the TTR gene, and more particularly one or more    pathogenic G-to-A or C-to-T mutations/SNPs described above.    Alpha-1-antitrypsin Deficiency

In some embodiments, the methods, systems, and compositions describedherein are used to correct one or more pathogenic G-to-A or C-to-Tmutations/SNPs associated with Alpha-1-antitrypsin deficiency. In someembodiment, the pathogenic mutations/SNPs are present in at least theSERPINA1 gene, including at least the followings:

-   NM_000295.4(SERPINA1):c.538C>T (p.Gln180Ter)-   NM_001127701.1(SERPINA1):c.1178C>T (p.Pro393Leu)-   NM_001127701.1(SERPINA1):c.230C>T (p.Ser77Phe)-   NM_001127701.1(SERPINA1):c.1096G>A (p.Glu366Lys)-   NM_000295.4(SERPINA1):c.1177C>T (p.Pro393Ser)    See Table A. Accordingly, an aspect of the invention relates to a    method for treating or preventing Alpha-1-antitrypsin deficiency by    correcting one or more pathogenic G-to-A or C-to-T mutations/SNPs,    particularly one or more pathogenic G-to-A or C-to-T mutations/SNPs    present in the SERPINA1 gene, and more particularly one or more    pathogenic G-to-A or C-to-T mutations/SNPs described above.

Wilson's Disease

In some embodiments, the methods, systems, and compositions describedherein are used to correct one or more pathogenic G-to-A or C-to-Tmutations/SNPs associated with Wilson's disease. In some embodiment, thepathogenic mutations/SNPs are present in at least the ATP7B gene,including at least the followings:

-   NM_000053.3(ATP7B):c.2293G>A (p.Asp765Asn)-   NM_000053.3(ATP7B):c.3955C>T (p.Arg1319Ter)-   NM_000053.3(ATP7B):c.2865+1G>A-   NM_000053.3(ATP7B):c.3796G>A (p.Gly1266Arg)-   NM_000053.3(ATP7B):c.2621C>T (p.Ala874Val)-   NM_000053.3(ATP7B):c.2071G>A (p.Gly691Arg)-   NM_000053.3(ATP7B):c.2128G>A (p.Gly710Ser)-   NM_000053.3(ATP7B):c.2336G>A (p.Trp779Ter)-   NM_000053.3(ATP7B):c.4021G>A (p.Gly1341Ser)-   NM_000053.3(ATP7B):c.3182G>A (p.Gly1061Glu)-   NM_000053.3(ATP7B):c.4114C>T (p.Gln1372Ter)-   NM_000053.3(ATP7B):c.1708-1G>A-   NM_000053.3(ATP7B):c.865C>T (p.Gln289Ter)-   NM_000053.3(ATP7B):c.2930C>T (p.Thr977Met)-   NM_000053.3(ATP7B):c.3659C>T (p.Thr1220Met)-   NM_000053.3(ATP7B):c.2605G>A (p.Gly869Arg)-   NM_000053.3(ATP7B):c.2975C>T (p.Pro992Leu)-   NM_000053.3(ATP7B):c.2519C>T (p.Pro840Leu)-   NM_000053.3(ATP7B):c.2906G>A (p.Arg969Gln)    See Table A. Accordingly, an aspect of the invention relates to a    method for treating or preventing Wilson's disease by correcting one    or more pathogenic G-to-A or C-to-T mutations/SNPs, particularly one    or more pathogenic G-to-A or C-to-T mutations/SNPs present in the    ATP7B gene, and more particularly one or more pathogenic G-to-A or    C-to-T mutations/SNPs described above.

Phenylketonuria

In some embodiments, the methods, systems, and compositions describedherein are used to correct one or more pathogenic G-to-A or C-to-Tmutations/SNPs associated with Phenylketonuria. In some embodiment, thepathogenic mutations/SNPs are present in at least the PAH gene,including at least the followings:

-   NM_000277.1(PAH):c.1315+1G>A-   NM_000277.1(PAH):c.1222C>T (p.Arg408Trp)-   NM_000277.1(PAH):c.838G>A (p.Glu280Lys)-   NM_000277.1(PAH):c.331C>T (p.Arg111Ter)-   NM_000277.1(PAH):c.782G>A (p.Arg261Gln)-   NM_000277.1(PAH):c.754C>T (p.Arg252Trp)-   NM_000277.1(PAH):c.473G>A (p.Arg158Gln)-   NM_000277.1(PAH):c.727C>T (p.Arg243Ter)-   NM_000277.1(PAH):c.842C>T (p.Pro281Leu)-   NM_000277.1(PAH):c.728G>A (p.Arg243Gln)-   NM_000277.1(PAH):c.1066-11G>A-   NM_000277.1(PAH):c.781C>T (p.Arg261Ter)-   NM_000277.1(PAH):c.1223G>A (p.Arg408Gln)-   NM_000277.1(PAH):c.1162G>A (p.Val388Met)-   NM_000277.1(PAH):c.1066-3C>T-   NM_000277.1(PAH):c.1208C>T (p.Ala403Val)-   NM_000277.1(PAH):c.890G>A (p.Arg297His)-   NM_000277.1(PAH):c.926C>T (p.Ala309Val)-   NM_000277.1(PAH):c.441+1G>A-   NM_000277.1(PAH):c.526C>T (p.Arg176Ter)-   NM_000277.1(PAH):c.688G>A (p.Val230Ile)-   NM_000277.1(PAH):c.721C>T (p.Arg241Cys)-   NM_000277.1(PAH):c.745C>T (p.Leu249Phe)-   NM_000277.1(PAH):c.442-1G>A-   NM_000277.1(PAH):c.842+1G>A-   NM_000277.1(PAH):c.776C>T (p.Ala259Val)-   NM_000277.1(PAH):c.1200-1G>A-   NM_000277.1(PAH):c.912+1G>A-   NM_000277.1(PAH):c.1065+1G>A-   NM_000277.1(PAH):c.472C>T (p.Arg158Trp)-   NM_000277.1(PAH):c.755G>A (p.Arg252Gln)-   NM_000277.1(PAH):c.809G>A (p.Arg270Lys)    See Table A. Accordingly, an aspect of the invention relates to a    method for treating or preventing Phenylketonuria by correcting one    or more pathogenic G-to-A or C-to-T mutations/SNPs, particularly one    or more pathogenic G-to-A or C-to-T mutations/SNPs present in the    PAH gene, and more particularly one or more pathogenic G-to-A or    C-to-T mutations/SNPs described above.

Kidney Diseases

Pathogenic G-to-A or C-to-T mutations/SNPs associated with variouskidney diseases are reported in the ClinVar database and disclosed inTable A, including but not limited to Autosomal recessive polycystickidney disease and Renal carnitine transport defect. Accordingly, anaspect of the invention relates to a method for correcting one or morepathogenic G-to-A or C-to-T mutations/SNPs associated with any of thesediseases, as discussed below.

Autosomal Recessive Polycystic Kidney Disease

In some embodiments, the methods, systems, and compositions describedherein are used to correct one or more pathogenic G-to-A or C-to-Tmutations/SNPs associated with Autosomal recessive polycystic kidneydisease. In some embodiment, the pathogenic mutations/SNPs are presentin at least the PKHD1 gene, including at least the followings:

-   NM_138694.3(PKHD1):c.10444C>T (p.Arg3482Cys)-   NM_138694.3(PKHD1):c.9319C>T (p.Arg3107Ter)-   NM_138694.3(PKHD1):c.1480C>T (p.Arg494Ter)-   NM_138694.3(PKHD1):c.707+1G>A-   NM_138694.3(PKHD1):c.1486C>T (p.Arg496Ter)-   NM_138694.3(PKHD1):c.8303-1G>A-   NM_138694.3(PKHD1):c.2854G>A (p.Gly952Arg)-   NM_138694.3(PKHD1):c.7194G>A (p.Trp2398Ter)-   NM_138694.3(PKHD1):c.10219C>T (p.Gln3407Ter)-   NM_138694.3(PKHD1):c.107C>T (p.Thr36Met)-   NM_138694.3(PKHD1):c.8824C>T (p.Arg2942Ter)-   NM_138694.3(PKHD1):c.982C>T (p.Arg328Ter)-   NM_138694.3(PKHD1):c.4870C>T (p.Arg1624Trp)-   NM_138694.3(PKHD1):c.1602+1G>A-   NM_138694.3(PKHD1):c.1694-1G>A-   NM_138694.3(PKHD1):c.2341C>T (p.Arg781Ter)-   NM_138694.3(PKHD1):c.2407+1G>A-   NM_138694.3(PKHD1):c.2452C>T (p.Gln818Ter)-   NM_138694.3(PKHD1):c.5236+1G>A-   NM_138694.3(PKHD1):c.6499C>T (p.Gln2167Ter)-   NM_138694.3(PKHD1):c.2725C>T (p.Arg909Ter)-   NM_138694.3(PKHD1):c.370C>T (p.Arg124Ter)-   NM_138694.3(PKHD1):c.2810G>A (p.Trp937Ter)    See Table A. Accordingly, an aspect of the invention relates to a    method for treating or preventing Autosomal recessive polycystic    kidney disease by correcting one or more pathogenic G-to-A or C-to-T    mutations/SNPs, particularly one or more pathogenic G-to-A or C-to-T    mutations/SNPs present in the PKHD1 gene, and more particularly one    or more pathogenic G-to-A or C-to-T mutations/SNPs described above.

Renal Carnitine Transport Defect

In some embodiments, the methods, systems, and compositions describedherein are used to correct one or more pathogenic G-to-A or C-to-Tmutations/SNPs associated with Renal carnitine transport defect. In someembodiment, the pathogenic mutations/SNPs are present in at least theSLC22A5 gene, including at least the followings:

-   NM_003060.3(SLC22A5):c.760C>T (p.Arg254Ter)-   NM_003060.3(SLC22A5):c.396G>A (p.Trp132Ter)-   NM_003060.3(SLC22A5):c.844C>T (p.Arg282Ter)-   NM_003060.3(SLC22A5):c.505C>T (p.Arg169Trp)-   NM_003060.3(SLC22A5):c.1319C>T (p.Thr440Met)-   NM_003060.3(SLC22A5):c.1195C>T (p.Arg399Trp)-   NM_003060.3(SLC22A5):c.695C>T (p.Thr232Met)-   NM_003060.3(SLC22A5):c.845G>A (p.Arg282Gln)-   NM_003060.3(SLC22A5):c.1193C>T (p.Pro398Leu)-   NM_003060.3(SLC22A5):c.1463G>A (p.Arg488His)-   NM_003060.3(SLC22A5):c.338G>A (p.Cys113Tyr)-   NM_003060.3(SLC22A5):c.136C>T (p.Pro46Ser)-   NM_003060.3(SLC22A5):c.506G>A (p.Arg169Gln)    See Table A. Accordingly, an aspect of the invention relates to a    method for treating or preventing Renal carnitine transport defect    by correcting one or more pathogenic G-to-A or C-to-T    mutations/SNPs, particularly one or more pathogenic G-to-A or C-to-T    mutations/SNPs present in the SLC22A5 gene, and more particularly    one or more pathogenic G-to-A or C-to-T mutations/SNPs described    above.

Muscle Diseases

Pathogenic G-to-A or C-to-T mutations/SNPs associated with variousmuscle diseases are reported in the ClinVar database and disclosed inTable A, including but not limited to Duchenne muscular dystrophy,Becker muscular dystrophy, Limb-girdle muscular dystrophy,Emery-Dreifuss muscular dystrophy, and Facioscapulohumeral musculardystrophy. Accordingly, an aspect of the invention relates to a methodfor correcting one or more pathogenic G-to-A or C-to-T mutations/SNPsassociated with any of these diseases, as discussed below.

Duchenne Muscular Dystrophy

In some embodiments, the methods, systems, and compositions describedherein are used to correct one or more pathogenic G-to-A or C-to-Tmutations/SNPs associated with Duchenne muscular dystrophy. In someembodiment, the pathogenic mutations/SNPs are present in at least theDMD gene, including at least the followings:

-   NM_004006.2(DMD):c.2797C>T (p.Gln933Ter)-   NM_004006.2(DMD):c.4870C>T (p.Gln1624Ter)-   NM_004006.2(DMD):c.5551C>T (p.Gln1851Ter)-   NM_004006.2(DMD):c.3188G>A (p.Trp1063Ter)-   NM_004006.2(DMD):c.8357G>A (p.Trp2786Ter)-   NM_004006.2(DMD):c.7817G>A (p.Trp2606Ter)-   NM_004006.2(DMD):c.7755G>A (p.Trp2585Ter)-   NM_004006.2(DMD):c.5917C>T (p.Gln1973Ter)-   NM_004006.2(DMD):c.5641C>T (p.Gln1881Ter)-   NM_004006.2(DMD):c.5131C>T (p.Gln1711Ter)-   NM_004006.2(DMD):c.4240C>T (p.Gln1414Ter)-   NM_004006.2(DMD):c.3427C>T (p.Gln1143Ter)-   NM_004006.2(DMD):c.2407C>T (p.Gln803Ter)-   NM_004006.2(DMD):c.2368C>T (p.Gln790Ter)-   NM_004006.2(DMD):c.1683G>A (p.Trp561Ter)-   NM_004006.2(DMD):c.1663C>T (p.Gln555Ter)-   NM_004006.2(DMD):c.1388G>A (p.Trp463Ter)-   NM_004006.2(DMD):c.1331+1G>A-   NM_004006.2(DMD):c.1324C>T (p.Gln442Ter)-   NM_004006.2(DMD):c.355C>T (p.Gln119Ter)-   NM_004006.2(DMD):c.94-1G>A-   NM_004006.2(DMD):c.5506C>T (p.Gln1836Ter)-   NM_004006.2(DMD):c.1504C>T (p.Gln502Ter)-   NM_004006.2(DMD):c.5032C>T (p.Gln1678Ter)-   NM_004006.2(DMD):c.457C>T (p.Gln153Ter)-   NM_004006.2(DMD):c.1594C>T (p.Gln532Ter)-   NM_004006.2(DMD):c.1150-1G>A-   NM_004006.2(DMD):c.6223C>T (p.Gln2075Ter)-   NM_004006.2(DMD):c.3747G>A (p.Trp1249Ter)-   NM_004006.2(DMD):c.2861G>A (p.Trp954Ter)-   NM_004006.2(DMD):c.9563+1G>A-   NM_004006.2(DMD):c.4483C>T (p.Gln1495Ter)-   NM_004006.2(DMD):c.4312C>T (p.Gln1438Ter)-   NM_004006.2(DMD):c.8209C>T (p.Gln2737Ter)-   NM_004006.2(DMD):c.4071+1G>A-   NM_004006.2(DMD):c.2665C>T (p.Arg889Ter)-   NM_004006.2(DMD):c.2202G>A (p.Trp734Ter)-   NM_004006.2(DMD):c.2077C>T (p.Gln693Ter)-   NM_004006.2(DMD):c.1653G>A (p.Trp551Ter)-   NM_004006.2(DMD):c.1061G>A (p.Trp354Ter)-   NM_004006.2(DMD):c.8914C>T (p.Gln2972Ter)-   NM_004006.2(DMD):c.6118-1G>A-   NM_004006.2(DMD):c.4729C>T (p.Arg1577Ter)    See Table A. Accordingly, an aspect of the invention relates to a    method for treating or preventing Duchenne muscular dystrophy by    correcting one or more pathogenic G-to-A or C-to-T mutations/SNPs,    particularly one or more pathogenic G-to-A or C-to-T mutations/SNPs    present in the DMD gene, and more particularly one or more    pathogenic G-to-A or C-to-T mutations/SNPs described above.

Becker Muscular Dystrophy

In some embodiments, the methods, systems, and compositions describedherein are used to correct one or more pathogenic G-to-A or C-to-Tmutations/SNPs associated with Becker muscular dystrophy. In someembodiment, the pathogenic mutations/SNPs are present in at least theDMD gene, including at least the followings:

-   NM_004006.2(DMD):c.3413G>A (p.Trp1138Ter)-   NM_004006.2(DMD):c.358-1G>A-   NM_004006.2(DMD):c.10108C>T (p.Arg3370Ter)-   NM_004006.2(DMD):c.6373C>T (p.Gln2125Ter)-   NM_004006.2(DMD):c.9568C>T (p.Arg3190Ter)-   NM_004006.2(DMD):c.8713C>T (p.Arg2905Ter)-   NM_004006.2(DMD):c.1615C>T (p.Arg539Ter)-   NM_004006.2(DMD):c.3151C>T (p.Arg1051Ter)-   NM_004006.2(DMD):c.3432+1G>A-   NM_004006.2(DMD):c.5287C>T (p.Arg1763 Ter)-   NM_004006.2(DMD):c.5530C>T (p.Arg1844Ter)-   NM_004006.2(DMD):c.8608C>T (p.Arg2870Ter)-   NM_004006.2(DMD):c.8656C>T (p.Gln2886Ter)-   NM_004006.2(DMD):c.8944C>T (p.Arg2982Ter)-   NM_004006.2(DMD):c.5899C>T (p.Arg1967Ter)-   NM_004006.2(DMD):c.10033C>T (p.Arg3345Ter)-   NM_004006.2(DMD):c.10086+1G>A-   NM_004019.2(DMD):c.1020G>A (p.Thr340=)-   NM_004006.2(DMD):c.1261C>T (p.Gln421Ter)-   NM_004006.2(DMD):c.1465C>T (p.Gln489Ter)-   NM_004006.2(DMD):c.1990C>T (p.Gln664Ter)-   NM_004006.2(DMD):c.2032C>T (p.Gln678Ter)-   NM_004006.2(DMD):c.2332C>T (p.Gln778Ter)-   NM_004006.2(DMD):c.2419C>T (p.Gln807Ter)-   NM_004006.2(DMD):c.2650C>T (p.Gln884Ter)-   NM_004006.2(DMD):c.2804-1G>A-   NM_004006.2(DMD):c.3276+1G>A-   NM_004006.2(DMD):c.3295C>T (p.Gln1099Ter)-   NM_004006.2(DMD):c.336G>A (p.Trp112Ter)-   NM_004006.2(DMD):c.3580C>T (p.Gln1194Ter)-   NM_004006.2(DMD):c.4117C>T (p.Gln1373Ter)-   NM_004006.2(DMD):c.649+1G>A-   NM_004006.2(DMD):c.6906G>A (p.Trp2302Ter)-   NM_004006.2(DMD):c.7189C>T (p.Gln2397Ter)-   NM_004006.2(DMD):c.7309+1G>A-   NM_004006.2(DMD):c.7657C>T (p.Arg2553Ter)-   NM_004006.2(DMD):c.7682G>A (p.Trp2561Ter)-   NM_004006.2(DMD):c.7683G>A (p.Trp2561Ter)-   NM_004006.2(DMD):c.7894C>T (p.Gln2632Ter)-   NM_004006.2(DMD):c.9361+1G>A-   NM_004006.2(DMD):c.9564-1G>A-   NM_004006.2(DMD):c.2956C>T (p.Gln986Ter)-   NM_004006.2(DMD):c.883C>T (p.Arg295Ter)-   NM_004006.2(DMD):c.31+36947G>A-   NM_004006.2(DMD):c.10279C>T (p.Gln3427Ter)-   NM_004006.2(DMD):c.433C>T (p.Arg145Ter)-   NM_004006.2(DMD):c.9G>A (p.Trp3Ter)-   NM_004006.2(DMD):c.10171C>T (p.Arg3391Ter)-   NM_004006.2(DMD):c.583C>T (p.Arg195Ter)-   NM_004006.2(DMD):c.9337C>T (p.Arg3113Ter)-   NM_004006.2(DMD):c.8038C>T (p.Arg2680Ter)-   NM_004006.2(DMD):c.1812+1G>A-   NM_004006.2(DMD):c.1093C>T (p.Gln365Ter)-   NM_004006.2(DMD):c.1704+1G>A-   NM_004006.2(DMD):c.1912C>T (p.Gln638Ter)-   NM_004006.2(DMD):c.133C>T (p.Gln45Ter)-   NM_004006.2(DMD):c.5868G>A (p.Trp1956Ter)-   NM_004006.2(DMD):c.565C>T (p.Gln189Ter)-   NM_004006.2(DMD):c.5089C>T (p.Gln1697Ter)-   NM_004006.2(DMD):c.2512C>T (p.Gln838Ter)-   NM_004006.2(DMD):c.10477C>T (p.Gln3493Ter)-   NM_004006.2(DMD):c.93+1G>A-   NM_004006.2(DMD):c.4174C>T (p.Gln1392Ter)-   NM_004006.2(DMD):c.3940C>T (p.Arg1314Ter) See Table A. Accordingly,    an aspect of the invention relates to a method for treating or    preventing Becker muscular dystrophy by correcting one or more    pathogenic G-to-A or C-to-T mutations/SNPs, particularly one or more    pathogenic G-to-A or C-to-T mutations/SNPs present in the DMD gene,    and more particularly one or more pathogenic G-to-A or C-to-T    mutations/SNPs described above.

Limb-Girdle Muscular Dystrophy

In some embodiments, the methods, systems, and compositions describedherein are used to correct one or more pathogenic G-to-A or C-to-Tmutations/SNPs associated with Limb-girdle muscular dystrophy. In someembodiment, the pathogenic mutations/SNPs are present in at least onegene selected from SGCB, MYOT, LMNA, CAPN3, DYSF, SGCA, TTN, ANO5,TRAPPC11, LMNA, POMT1, and FKRP, including at least the followings:

-   NM_000232.4(SGCB):c.31C>T (p.Gln11Ter)-   NM_006790.2(MYOT):c.164C>T (p.Ser55Phe)-   NM_006790.2(MYOT):c.170C>T (p.Thr57Ile)-   NM_170707.3(LMNA):c.1488+1G>A-   NM_170707.3(LMNA):c.1609-1G>A-   NM_000070.2(CAPN3):c.1715G>A (p.Arg572Gln)-   NM_000070.2(CAPN3):c.2243G>A (p.Arg748Gln)-   NM_000070.2(CAPN3):c.145C>T (p.Arg49Cys)-   NM_000070.2(CAPN3):c.1319G>A (p.Arg440Gln)-   NM_000070.2(CAPN3):c.1343G>A (p.Arg448His)-   NM_000070.2(CAPN3):c.1465C>T (p.Arg489Trp)-   NM_000070.2(CAPN3):c.1714C>T (p.Arg572Trp)-   NM_000070.2(CAPN3):c.2306G>A (p.Arg769Gln)-   NM_000070.2(CAPN3):c.133G>A (p.Ala45Thr)-   NM_000070.2(CAPN3):c.499-1G>A-   NM_000070.2(CAPN3):c.439C>T (p.Arg147Ter)-   NM_000070.2(CAPN3):c.1063C>T (p.Arg355Trp)-   NM_000070.2(CAPN3):c.1250C>T (p.Thr417Met)-   NM_000070.2(CAPN3):c.245C>T (p.Pro82Leu)-   NM_000070.2(CAPN3):c.2242C>T (p.Arg748Ter)-   NM_000070.2(CAPN3):c.1318C>T (p.Arg440Trp)-   NM_000070.2(CAPN3):c.1333G>A (p.Gly445Arg)-   NM_000070.2(CAPN3):c.1957C>T (p.Gln653Ter)-   NM_000070.2(CAPN3):c.1801-1G>A-   NM_000070.2(CAPN3):c.2263+1G>A-   NM_000070.2(CAPN3):c.956C>T (p.Pro319Leu)-   NM_000070.2(CAPN3):c.1468C>T (p.Arg490Trp)-   NM_000070.2(CAPN3):c.802-9G>A-   NM_000070.2(CAPN3):c.1342C>T (p.Arg448Cys)-   NM_000070.2(CAPN3):c.1303G>A (p.Glu435Lys)-   NM_000070.2(CAPN3):c.1993-1G>A-   NM_003494.3(DYSF):c.3113G>A (p.Arg1038Gln)-   NM_001130987.1(DYSF):c.5174+1G>A-   NM_001130987.1(DYSF):c.159G>A (p.Trp53Ter)-   NM_001130987.1(DYSF):c.2929C>T (p.Arg977Trp)-   NM_001130987.1(DYSF):c.4282C>T (p.Gln1428Ter)-   NM_001130987.1(DYSF):c.1577-1G>A-   NM_003494.3(DYSF):c.5529G>A (p.Trp1843Ter)-   NM_001130987.1(DYSF):c.1576+1G>A-   NM_001130987.1(DYSF):c.4462C>T (p.Gln1488Ter)-   NM_003494.3(DYSF):c.5429G>A (p.Arg1810Lys)-   NM_003494.3(DYSF):c.5077C>T (p.Arg1693Trp)-   NM_001130978.1(DYSF):c.1813C>T (p.Gln605Ter)-   NM_003494.3(DYSF):c.3230G>A (p.Trp1077Ter)-   NM_003494.3(DYSF):c.265C>T (p.Arg89Ter)-   NM_003494.3(DYSF):c.4434G>A (p.Trp1478Ter)-   NM_003494.3(DYSF):c.3478C>T (p.Gln1160Ter)-   NM_001130987.1(DYSF):c.1372G>A (p.Gly458Arg)-   NM_003494.3(DYSF):c.4090C>T (p.Gln1364Ter)-   NM_001130987.1(DYSF):c.2409+1G>A-   NM_003494.3(DYSF):c.1708C>T (p.Gln570Ter)-   NM_003494.3(DYSF):c.1956G>A (p.Trp652Ter)-   NM_001130987.1(DYSF):c.5004-1G>A-   NM_003494.3(DYSF):c.331C>T (p.Gln111Ter)-   NM_001130978.1(DYSF):c.5776C>T (p.Arg1926Ter)-   NM_003494.3(DYSF):c.6124C>T (p.Arg2042Cys)-   NM_003494.3(DYSF):c.2643+1G>A-   NM_003494.3(DYSF):c.4253G>A (p.Gly1418Asp)-   NM_003494.3(DYSF):c.610C>T (p.Arg204Ter)-   NM_003494.3(DYSF):c.1834C>T (p.Gln612Ter)-   NM_003494.3(DYSF):c.5668-7G>A-   NM_001130978.1(DYSF):c.3137G>A (p.Arg1046His)-   NM_003494.3(DYSF):c.1053+1G>A-   NM_003494.3(DYSF):c.1398-1G>A-   NM_003494.3(DYSF):c.1481-1G>A-   NM_003494.3(DYSF):c.2311C>T (p.Gln771Ter)-   NM_003494.3(DYSF):c.2869C>T (p.Gln957Ter)-   NM_003494.3(DYSF):c.4756C>T (p.Arg1586Ter)-   NM_003494.3(DYSF):c.5509G>A (p.Asp1837Asn)-   NM_003494.3(DYSF):c.5644C>T (p.Gln1882Ter)-   NM_003494.3(DYSF):c.5946+1G>A-   NM_003494.3(DYSF):c.937+1G>A-   NM_003494.3(DYSF):c.5266C>T (p.Gln1756Ter)-   NM_003494.3(DYSF):c.3832C>T (p.Gln1278Ter)-   NM_003494.3(DYSF):c.5525+1G>A-   NM_003494.3(DYSF):c.3112C>T (p.Arg1038Ter)-   NM_000023.3(SGCA):c.293G>A (p.Arg98His)-   NM_000023.3(SGCA):c.850C>T (p.Arg284Cys)-   NM_000023.3(SGCA):c.403C>T (p.Gln135Ter)-   NM_000023.3(SGCA):c.409G>A (p.Glu137Lys)-   NM_000023.3(SGCA):c.747+1G>A-   NM_000023.3(SGCA):c.229C>T (p.Arg77Cys)-   NM_000023.3(SGCA):c.101G>A (p.Arg34His)-   NM_000023.3(SGCA):c.739G>A (p.Val247Met)-   NM_001256850.1(TTN):c.87394C>T (p.Arg29132Ter)-   NM_213599.2(ANO5):c.762+1G>A-   NM_213599.2(ANO5):c.1213C>T (p.Gln405Ter)-   NM_213599.2(ANO5):c.1639C>T (p.Arg547Ter)-   NM_213599.2(ANO5):c.1406G>A (p.Trp469Ter)-   NM_213599.2(ANO5):c.1210C>T (p.Arg404Ter)-   NM_213599.2(ANO5):c.2272C>T (p.Arg758Cys)-   NM_213599.2(ANO5):c.41-1G>A-   NM_213599.2(ANO5):c.172C>T (p.Arg58Trp)-   NM_213599.2(ANO5):c.1898+1G>A-   NM_021942.5(TRAPPC11):c.1287+5G>A-   NM_170707.3(LMNA):c.1608+1G>A-   NM_007171.3(POMT1):c.1864C>T (p.Arg622Ter)-   NM_024301.4(FKRP):c.313C>T (p.Gln105Ter)    See Table A. Accordingly, an aspect of the invention relates to a    method for treating or preventing Limb-girdle muscular dystrophy by    correcting one or more pathogenic G-to-A or C-to-T mutations/SNPs,    particularly one or more pathogenic G-to-A or C-to-T mutations/SNPs    present in at least one gene selected from SGCB, MYOT, LMNA, CAPN3,    DYSF, SGCA, TTN, ANO5, TRAPPC11, LMNA, POMT1, and FKRP, and more    particularly one or more pathogenic G-to-A or C-to-T mutations/SNPs    described above.

Emery-Dreifuss Muscular Dystrophy

In some embodiments, the methods, systems, and compositions describedherein are used to correct one or more pathogenic G-to-A or C-to-Tmutations/SNPs associated with Emery-Dreifuss muscular dystrophy. Insome embodiment, the pathogenic mutations/SNPs are present in at leastthe EMD or SYNE1 gene, including at least the followings:

-   NM_000117.2(EMD):c.3G>A (p.Met1Ile)-   NM_033071.3(SYNE1):c.11908C>T (p.Arg3970Ter)-   NM_033071.3(SYNE1):c.21721C>T (p.Gln7241Ter)-   NM_000117.2(EMD):c.130C>T (p.Gln44Ter)    See Table A. Accordingly, an aspect of the invention relates to a    method for treating or preventing Emery-Dreifuss muscular dystrophy    by correcting one or more pathogenic G-to-A or C-to-T    mutations/SNPs, particularly one or more pathogenic G-to-A or C-to-T    mutations/SNPs present in the EMD or SYNE1 gene, and more    particularly one or more pathogenic G-to-A or C-to-T mutations/SNPs    described above.

Facioscapulohumeral Muscular Dystrophy

In some embodiments, the methods, systems, and compositions describedherein are used to correct one or more pathogenic G-to-A or C-to-Tmutations/SNPs associated with Facioscapulohumeral muscular dystrophy.In some embodiment, the pathogenic mutations/SNPs are present in atleast the SMCHD1 gene, including at least the followings:

-   NM_015295.2(SMCHD1):c.3801+1G>A-   NM_015295.2(SMCHD1):c.1843-1G>A    See Table A. Accordingly, an aspect of the invention relates to a    method for treating or preventing Facioscapulohumeral muscular    dystrophy by correcting one or more pathogenic G-to-A or C-to-T    mutations/SNPs, particularly one or more pathogenic G-to-A or C-to-T    mutations/SNPs present in the SMCHD1 gene, and more particularly one    or more pathogenic G-to-A or C-to-T mutations/SNPs described above.

Inborn Errors of Metabolism (IEM)

Pathogenic G-to-A or C-to-T mutations/SNPs associated with various IEMsare reported in the ClinVar database and disclosed in Table A, includingbut not limited to Primary hyperoxaluria type 1, Argininosuccinate lyasedeficiency, Ornithine carbamoyltransferase deficiency, and Maple syrupurine disease. Accordingly, an aspect of the invention relates to amethod for correcting one or more pathogenic G-to-A or C-to-Tmutations/SNPs associated with any of these diseases, as discussedbelow.

Primary Hyperoxaluria Type 1

In some embodiments, the methods, systems, and compositions describedherein are used to correct one or more pathogenic G-to-A or C-to-Tmutations/SNPs associated with Primary hyperoxaluria type 1. In someembodiment, the pathogenic mutations/SNPs are present in at least theAGXT gene, including at least the followings:

-   NM_000030.2(AGXT):c.245G>A (p.Gly82Glu)-   NM_000030.2(AGXT):c.698G>A (p.Arg233His)-   NM_000030.2(AGXT):c.466G>A (p.Gly156Arg)-   NM_000030.2(AGXT):c.106C>T (p.Arg36Cys)-   NM_000030.2(AGXT):c.346G>A (p.Gly116Arg)-   NM_000030.2(AGXT):c.568G>A (p.Gly190Arg)-   NM_000030.2(AGXT):c.653C>T (p.Ser218Leu)-   NM_000030.2(AGXT):c.737G>A (p.Trp246Ter)-   NM_000030.2(AGXT):c.1049G>A (p.Gly350Asp)-   NM_000030.2(AGXT):c.473C>T (p.Ser158Leu)-   NM_000030.2(AGXT):c.907C>T (p.Gln303Ter)-   NM_000030.2(AGXT):c.996G>A (p.Trp332Ter)-   NM_000030.2(AGXT):c.508G>A (p.Gly170Arg)    See Table A. Accordingly, an aspect of the invention relates to a    method for treating or preventing Primary hyperoxaluria type 1 by    correcting one or more pathogenic G-to-A or C-to-T mutations/SNPs,    particularly one or more pathogenic G-to-A or C-to-T mutations/SNPs    present in the AGXT gene, and more particularly one or more    pathogenic G-to-A or C-to-T mutations/SNPs described above.

Argininosuccinate Lyase Deficiency

In some embodiments, the methods, systems, and compositions describedherein are used to correct one or more pathogenic G-to-A or C-to-Tmutations/SNPs associated with Argininosuccinate lyase deficiency. Insome embodiment, the pathogenic mutations/SNPs are present in at leastthe ASL gene, including at least the followings:

-   NM_001024943.1(ASL):c.1153C>T (p.Arg385Cys)-   NM_000048.3(ASL):c.532G>A (p.Val178Met)-   NM_000048.3(ASL):c.545G>A (p.Arg182Gln)-   NM_000048.3(ASL):c.175G>A (p.Glu59Lys)-   NM_000048.3(ASL):c.718+5G>A-   NM_000048.3(ASL):c.889C>T (p.Arg297Trp)-   NM_000048.3(ASL):c.1360C>T (p.Gln454Ter)-   NM_000048.3(ASL):c.1060C>T (p.Gln354Ter)-   NM_000048.3(ASL):c.35G>A (p.Arg12Gln)-   NM_000048.3(ASL):c.446+1G>A-   NM_000048.3(ASL):c.544C>T (p.Arg182Ter)-   NM_000048.3(ASL):c.1135C>T (p.Arg379Cys)    See Table A. Accordingly, an aspect of the invention relates to a    method for treating or preventing Argininosuccinate lyase deficiency    by correcting one or more pathogenic G-to-A or C-to-T    mutations/SNPs, particularly one or more pathogenic G-to-A or C-to-T    mutations/SNPs present in the ASL gene, and more particularly one or    more pathogenic G-to-A or C-to-T mutations/SNPs described above.

Ornithine Carbamoyltransferase Deficiency

In some embodiments, the methods, systems, and compositions describedherein are used to correct one or more pathogenic G-to-A or C-to-Tmutations/SNPs associated with Ornithine carbamoyltransferasedeficiency. In some embodiment, the pathogenic mutations/SNPs arepresent in at least the OTC gene, including at least the followings:

-   NM_000531.5(OTC):c.119G>A (p.Arg40His)-   NM_000531.5(OTC):c.422G>A (p.Arg141Gln)-   NM_000531.5(OTC):c.829C>T (p.Arg277Trp)-   NM_000531.5(OTC):c.674C>T (p.Pro225Leu)    See Table A. Accordingly, an aspect of the invention relates to a    method for treating or preventing Ornithine carbamoyltransferase    deficiency by correcting one or more pathogenic G-to-A or C-to-T    mutations/SNPs, particularly one or more pathogenic G-to-A or C-to-T    mutations/SNPs present in the OTC gene, and more particularly one or    more pathogenic G-to-A or C-to-T mutations/SNPs described above.

Maple Syrup Urine Disease

In some embodiments, the methods, systems, and compositions describedherein are used to correct one or more pathogenic G-to-A or C-to-Tmutations/SNPs associated with Maple syrup urine disease. In someembodiment, the pathogenic mutations/SNPs are present in at least onegene selected from BCKDHA, BCKDHB, DBT, and DLD, including at least thefollowings:

-   NM_000709.3(BCKDHA):c.476G>A (p.Arg159Gln)-   NM_183050.3(BCKDHB):c.3G>A (p.Met1Ile)-   NM_183050.3(BCKDHB):c.554C>T (p.Pro185Leu)-   NM_001918.3(DBT):c.1033G>A (p.Gly345Arg)-   NM_000709.3(BCKDHA):c.940C>T (p.Arg314Ter)-   NM_000709.3(BCKDHA):c.793C>T (p.Arg265Trp)-   NM_000709.3(BCKDHA):c.868G>A (p.Gly290Arg)-   NM_000108.4(DLD):c.1123G>A (p.Glu375Lys)-   NM_000709.3(BCKDHA):c.1234G>A (p.Val412Met)-   NM_000709.3(BCKDHA):c.288+1G>A-   NM_000709.3(BCKDHA):c.979G>A (p.Glu327Lys)-   NM_001918.3(DBT):c.901C>T (p.Arg301Cys)-   NM_183050.3(BCKDHB):c.509G>A (p.Arg170His)-   NM_183050.3(BCKDHB):c.799C>T (p.Gln267Ter)-   NM_183050.3(BCKDHB):c.853C>T (p.Arg285Ter)-   NM_183050.3(BCKDHB):c.970C>T (p.Arg324Ter)-   NM_183050.3(BCKDHB):c.832G>A (p.Gly278Ser)-   NM_000709.3(BCKDHA):c.1036C>T (p.Arg346Cys)-   NM_000709.3(BCKDHA):c.288+9C>T-   NM_000709.3(BCKDHA):c.632C>T (p.Thr211Met)-   NM_000709.3(BCKDHA):c.659C>T (p.Ala220Val)-   NM_000709.3(BCKDHA):c.964C>T (p.Gln322Ter)-   NM_001918.3(DBT):c.1291C>T (p.Arg431Ter)-   NM_001918.3(DBT):c.251G>A (p.Trp84Ter)-   NM_001918.3(DBT):c.871C>T (p.Arg291Ter)-   NM_000056.4(BCKDHB):c.1016C>T (p.Ser339Leu)-   NM_000056.4(BCKDHB):c.344-1G>A-   NM_000056.4(BCKDHB):c.633+1G>A-   NM_000056.4(BCKDHB):c.952-1G>A    See Table A. Accordingly, an aspect of the invention relates to a    method for treating or preventing Maple syrup urine disease by    correcting one or more pathogenic G-to-A or C-to-T mutations/SNPs,    particularly one or more pathogenic G-to-A or C-to-T mutations/SNPs    present in at least one gene selected from BCKDHA, BCKDHB, DBT, and    DLD, and more particularly one or more pathogenic G-to-A or C-to-T    mutations/SNPs described above.

Cancer-Related Diseases

Pathogenic G-to-A or C-to-T mutations/SNPs associated with variouscancers and cancer-related diseases are reported in the ClinVar databaseand disclosed in Table A, including but not limited to Breast-OvarianCancer and Lynch syndrome. Accordingly, an aspect of the inventionrelates to a method for correcting one or more pathogenic G-to-A orC-to-T mutations/SNPs associated with any of these diseases, asdiscussed below.

Breast-Ovarian Cancer

In some embodiments, the methods, systems, and compositions describedherein are used to correct one or more pathogenic G-to-A or C-to-Tmutations/SNPs associated with Breast-Ovarian Cancer. In someembodiment, the pathogenic mutations/SNPs are present in at least theBRCA1or BRCA2 gene, including at least the followings:

-   NM_007294.3(BRCA1):c.5095C>T (p.Arg1699Trp)-   NM_000059.3(BRCA2):c.7558C>T (p.Arg2520Ter)-   NM_007294.3(BRCA1):c.2572C>T (p.Gln858Ter)-   NM_007294.3(BRCA1):c.3607C>T (p.Arg1203Ter)-   NM_007294.3(BRCA1):c.5503C>T (p.Arg1835Ter)-   NM_007294.3(BRCA1):c.2059C>T (p.Gln687Ter)-   NM_007294.3(BRCA1):c.4675+1G>A-   NM_007294.3(BRCA1):c.5251C>T (p.Arg1751Ter)-   NM_007294.3(BRCA1):c.5444G>A (p.Trp1815Ter)-   NM_000059.3(BRCA2):c.9318G>A (p.Trp3106Ter)-   NM_000059.3(BRCA2):c.9382C>T (p.Arg3128Ter)-   NM_000059.3(BRCA2):c.274C>T (p.Gln92Ter)-   NM_000059.3(BRCA2):c.6952C>T (p.Arg2318Ter)-   NM_007294.3(BRCA1):c.1687C>T (p.Gln563Ter)-   NM_007294.3(BRCA1):c.2599C>T (p.Gln867Ter)-   NM_007294.3(BRCA1):c.784C>T (p.Gln262Ter)-   NM_007294.3(BRCA1):c.280C>T (p.Gln94Ter)-   NM_007294.3(BRCA1):c.5542C>T (p.Gln1848Ter)-   NM_007294.3(BRCA1):c.5161C>T (p.Gln1721Ter)-   NM_007294.3(BRCA1):c.4573C>T (p.Gln1525Ter)-   NM_007294.3(BRCA1):c.4270C>T (p.Gln1424Ter)-   NM_007294.3(BRCA1):c.4225C>T (p.Gln1409Ter)-   NM_007294.3(BRCA1):c.4066C>T (p.Gln1356Ter)-   NM_007294.3(BRCA1):c.3679C>T (p.Gln1227Ter)-   NM_007294.3(BRCA1):c.1918C>T (p.Gln640Ter)-   NM_007294.3(BRCA1):c.963G>A (p.Trp321Ter)-   NM_007294.3(BRCA1):c.718C>T (p.Gln240Ter)-   NM_000059.3(BRCA2):c.9196C>T (p.Gln3066Ter)-   NM_000059.3(BRCA2):c.9154C>T (p.Arg3052Trp)-   NM_007294.3(BRCA1):c.3991C>T (p.Gln1331Ter)-   NM_007294.3(BRCA1):c.4097-1G>A-   NM_007294.3(BRCA1):c.1059G>A (p.Trp353Ter)-   NM_007294.3(BRCA1):c.1115G>A (p.Trp372Ter)-   NM_007294.3(BRCA1):c.1138C>T (p.Gln380Ter)-   NM_007294.3(BRCA1):c.1612C>T (p.Gln538Ter)-   NM_007294.3(BRCA1):c.1621C>T (p.Gln541Ter)-   NM_007294.3(BRCA1):c.1630C>T (p.Gln544Ter)-   NM_007294.3(BRCA1):c.178C>T (p.Gln60Ter)-   NM_007294.3(BRCA1):c.1969C>T (p.Gln657Ter)-   NM_007294.3(BRCA1):c.2275C>T (p.Gln759Ter)-   NM_007294.3(BRCA1):c.2410C>T (p.Gln804Ter)-   NM_007294.3(BRCA1):c.2869C>T (p.Gln957Ter)-   NM_007294.3(BRCA1):c.2923C>T (p.Gln975Ter)-   NM_007294.3(BRCA1):c.3268C>T (p.Gln1090Ter)-   NM_007294.3(BRCA1):c.3430C>T (p.Gln1144Ter)-   NM_007294.3(BRCA1):c.3544C>T (p.Gln1182Ter)-   NM_007294.3(BRCA1):c.4075C>T (p.Gln1359Ter)-   NM_007294.3(BRCA1):c.4201C>T (p.Gln1401Ter)-   NM_007294.3(BRCA1):c.4399C>T (p.Gln1467Ter)-   NM_007294.3(BRCA1):c.4552C>T (p.Gln1518Ter)-   NM_007294.3(BRCA1):c.5054C>T (p.Thr1685Ile)-   NM_007294.3(BRCA1):c.514C>T (p.Gln172Ter)-   NM_007294.3(BRCA1):c.5239C>T (p.Gln1747Ter)-   NM_007294.3(BRCA1):c.5266C>T (p.Gln1756Ter)-   NM_007294.3(BRCA1):c.5335C>T (p.Gln1779Ter)-   NM_007294.3(BRCA1):c.5345G>A (p.Trp1782Ter)-   NM_007294.3(BRCA1):c.5511G>A (p.Trp1837Ter)-   NM_007294.3(BRCA1):c.5536C>T (p.Gln1846Ter)-   NM_007294.3(BRCA1):c.55C>T (p.Gln19Ter)-   NM_007294.3(BRCA1):c.949C>T (p.Gln317Ter)-   NM_007294.3(BRCA1):c.928C>T (p.Gln310Ter)-   NM_007294.3(BRCA1):c.5117G>A (p.Gly1706Glu)-   NM_007294.3(BRCA1):c.5136G>A (p.Trp1712Ter)-   NM_007294.3(BRCA1):c.4327C>T (p.Arg1443Ter)-   NM_007294.3(BRCA1):c.1471C>T (p.Gln491Ter)-   NM_007294.3(BRCA1):c.1576C>T (p.Gln526Ter)-   NM_007294.3(BRCA1):c.160C>T (p.Gln54Ter)-   NM_007294.3(BRCA1):c.2683C>T (p.Gln895Ter)-   NM_007294.3(BRCA1):c.2761C>T (p.Gln921Ter)-   NM_007294.3(BRCA1):c.3895C>T (p.Gln1299Ter)-   NM_007294.3(BRCA1):c.4339C>T (p.Gln1447Ter)-   NM_007294.3(BRCA1):c.4372C>T (p.Gln1458Ter)-   NM_007294.3(BRCA1):c.5153G>A (p.Trp1718Ter)-   NM_007294.3(BRCA1):c.5445G>A (p.Trp1815Ter)-   NM_007294.3(BRCA1):c.5510G>A (p.Trp1837Ter)-   NM_007294.3(BRCA1):c.5346G>A (p.Trp1782Ter)-   NM_007294.3(BRCA1):c.1116G>A (p.Trp372Ter)-   NM_007294.3(BRCA1):c.1999C>T (p.Gln667Ter)-   NM_007294.3(BRCA1):c.4183C>T (p.Gln1395Ter)-   NM_007294.3(BRCA1):c.4810C>T (p.Gln1604Ter)-   NM_007294.3(BRCA1):c.850C>T (p.Gln284Ter)-   NM_007294.3(BRCA1):c.1058G>A (p.Trp353Ter)-   NM_007294.3(BRCA1):c.131G>A (p.Cys44Tyr)-   NM_007294.3(BRCA1):c.1600C>T (p.Gln534Ter)-   NM_007294.3(BRCA1):c.3286C>T (p.Gln1096Ter)-   NM_007294.3(BRCA1):c.3403C>T (p.Gln1135Ter)-   NM_007294.3(BRCA1):c.34C>T (p.Gln12Ter)-   NM_007294.3(BRCA1):c.4258C>T (p.Gln1420Ter)-   NM_007294.3(BRCA1):c.4609C>T (p.Gln1537Ter)-   NM_007294.3(BRCA1):c.5154G>A (p.Trp1718Ter)-   NM_007294.3(BRCA1):c.5431C>T (p.Gln1811Ter)-   NM_007294.3(BRCA1):c.241C>T (p.Gln81Ter)-   NM_007294.3(BRCA1):c.3331C>T (p.Gln1111Ter)-   NM_007294.3(BRCA1):c.3967C>T (p.Gln1323Ter)-   NM_007294.3(BRCA1):c.415C>T (p.Gln139Ter)-   NM_007294.3(BRCA1):c.505C>T (p.Gln169Ter)-   NM_007294.3(BRCA1):c.5194-12G>A-   NM_007294.3(BRCA1):c.5212G>A (p.Gly1738Arg)-   NM_007294.3(BRCA1):c.5332+1G>A-   NM_007294.3(BRCA1):c.1480C>T (p.Gln494Ter)-   NM_007294.3(BRCA1):c.2563C>T (p.Gln855Ter)-   NM_007294.3(BRCA1):c.1066C>T (p.Gln356Ter)-   NM_007294.3(BRCA1):c.3718C>T (p.Gln1240Ter)-   NM_007294.3(BRCA1):c.3817C>T (p.Gln1273Ter)-   NM_007294.3(BRCA1):c.3937C>T (p.Gln1313Ter)-   NM_007294.3(BRCA1):c.4357+1G>A-   NM_007294.3(BRCA1):c.5074+1G>A-   NM_007294.3(BRCA1):c.5277+1G>A-   NM_007294.3(BRCA1):c.2338C>T (p.Gln780Ter)-   NM_007294.3(BRCA1):c.3598C>T (p.Gln1200Ter)-   NM_007294.3(BRCA1):c.3841C>T (p.Gln1281Ter)-   NM_007294.3(BRCA1):c.4222C>T (p.Gln1408Ter)-   NM_007294.3(BRCA1):c.4524G>A (p.Trp1508Ter)-   NM_007294.3(BRCA1):c.5353C>T (p.Gln1785Ter)-   NM_007294.3(BRCA1):c.962G>A (p.Trp321Ter)-   NM_007294.3(BRCA1):c.220C>T (p.Gln74Ter)-   NM_007294.3(BRCA1):c.2713C>T (p.Gln905Ter)-   NM_007294.3(BRCA1):c.2800C>T (p.Gln934Ter)-   NM_007294.3(BRCA1):c.4612C>T (p.Gln1538Ter)-   NM_007294.3(BRCA1):c.3352C>T (p.Gln1118Ter)-   NM_007294.3(BRCA1):c.4834C>T (p.Gln1612Ter)-   NM_007294.3(BRCA1):c.4523G>A (p.Trp1508Ter)-   NM_007294.3(BRCA1):c.5135G>A (p.Trp1712Ter)-   NM_007294.3(BRCA1):c.1155G>A (p.Trp385Ter)-   NM_007294.3(BRCA1):c.4987-1G>A-   NM_000059.3(BRCA2):c.9573G>A (p.Trp3191Ter)-   NM_000059.3(BRCA2):c.1945C>T (p.Gln649Ter)-   NM_000059.3(BRCA2):c.217C>T (p.Gln73Ter)-   NM_000059.3(BRCA2):c.523C>T (p.Gln175Ter)-   NM_000059.3(BRCA2):c.2548C>T (p.Gln850Ter)-   NM_000059.3(BRCA2):c.2905C>T (p.Gln969Ter)-   NM_000059.3(BRCA2):c.4689G>A (p.Trp1563Ter)-   NM_000059.3(BRCA2):c.4972C>T (p.Gln1658Ter)-   NM_000059.3(BRCA2):c.1184G>A (p.Trp395Ter)-   NM_000059.3(BRCA2):c.2137C>T (p.Gln713Ter)-   NM_000059.3(BRCA2):c.3217C>T (p.Gln1073Ter)-   NM_000059.3(BRCA2):c.3523C>T (p.Gln1175Ter)-   NM_000059.3(BRCA2):c.4783C>T (p.Gln1595Ter)-   NM_000059.3(BRCA2):c.5800C>T (p.Gln1934Ter)-   NM_000059.3(BRCA2):c.6478C>T (p.Gln2160Ter)-   NM_000059.3(BRCA2):c.7033C>T (p.Gln2345Ter)-   NM_000059.3(BRCA2):c.7495C>T (p.Gln2499Ter)-   NM_000059.3(BRCA2):c.7501C>T (p.Gln2501Ter)-   NM_000059.3(BRCA2):c.7887G>A (p.Trp2629Ter)-   NM_000059.3(BRCA2):c.8910G>A (p.Trp2970Ter)-   NM_000059.3(BRCA2):c.9139C>T (p.Gln3047Ter)-   NM_000059.3(BRCA2):c.9739C>T (p.Gln3247Ter)-   NM_000059.3(BRCA2):c.582G>A (p.Trp194Ter)-   NM_000059.3(BRCA2):c.7963C>T (p.Gln2655Ter)-   NM_000059.3(BRCA2):c.8695C>T (p.Gln2899Ter)-   NM_000059.3(BRCA2):c.8869C>T (p.Gln2957Ter)-   NM_000059.3(BRCA2):c.1117C>T (p.Gln373Ter)-   NM_000059.3(BRCA2):c.1825C>T (p.Gln609Ter)-   NM_000059.3(BRCA2):c.2455C>T (p.Gln819Ter)-   NM_000059.3(BRCA2):c.2881C>T (p.Gln961Ter)-   NM_000059.3(BRCA2):c.3265C>T (p.Gln1089Ter)-   NM_000059.3(BRCA2):c.3283C>T (p.Gln1095Ter)-   NM_000059.3(BRCA2):c.3442C>T (p.Gln1148Ter)-   NM_000059.3(BRCA2):c.3871C>T (p.Gln1291Ter)-   NM_000059.3(BRCA2):c.439C>T (p.Gln147Ter)-   NM_000059.3(BRCA2):c.4525C>T (p.Gln1509Ter)-   NM_000059.3(BRCA2):c.475+1G>A-   NM_000059.3(BRCA2):c.5344C>T (p.Gln1782Ter)-   NM_000059.3(BRCA2):c.5404C>T (p.Gln1802Ter)-   NM_000059.3(BRCA2):c.5773C>T (p.Gln1925Ter)-   NM_000059.3(BRCA2):c.5992C>T (p.Gln1998Ter)-   NM_000059.3(BRCA2):c.6469C>T (p.Gln2157Ter)-   NM_000059.3(BRCA2):c.7261C>T (p.Gln2421Ter)-   NM_000059.3(BRCA2):c.7303C>T (p.Gln2435Ter)-   NM_000059.3(BRCA2):c.7471C>T (p.Gln2491Ter)-   NM_000059.3(BRCA2):c.7681C>T (p.Gln2561Ter)-   NM_000059.3(BRCA2):c.7738C>T (p.Gln2580Ter)-   NM_000059.3(BRCA2):c.7886G>A (p.Trp2629Ter)-   NM_000059.3(BRCA2):c.8140C>T (p.Gln2714Ter)-   NM_000059.3(BRCA2):c.8363G>A (p.Trp2788Ter)-   NM_000059.3(BRCA2):c.8572C>T (p.Gln2858Ter)-   NM_000059.3(BRCA2):c.8773C>T (p.Gln2925Ter)-   NM_000059.3(BRCA2):c.8821C>T (p.Gln2941Ter)-   NM_000059.3(BRCA2):c.9109C>T (p.Gln3037Ter)-   NM_000059.3(BRCA2):c.9317G>A (p.Trp3106Ter)-   NM_000059.3(BRCA2):c.9466C>T (p.Gln3156Ter)-   NM_000059.3(BRCA2):c.9572G>A (p.Trp3191Ter)-   NM_000059.3(BRCA2):c.8490G>A (p.Trp2830Ter)-   NM_000059.3(BRCA2):c.5980C>T (p.Gln1994Ter)-   NM_000059.3(BRCA2):c.7721G>A (p.Trp2574Ter)-   NM_000059.3(BRCA2):c.196C>T (p.Gln66Ter)-   NM_000059.3(BRCA2):c.7618-1G>A-   NM_000059.3(BRCA2):c.8489G>A (p.Trp2830Ter)-   NM_000059.3(BRCA2):c.7857G>A (p.Trp2619Ter)-   NM_000059.3(BRCA2):c.1261C>T (p.Gln421Ter)-   NM_000059.3(BRCA2):c.1456C>T (p.Gln486Ter)-   NM_000059.3(BRCA2):c.3319C>T (p.Gln1107Ter)-   NM_000059.3(BRCA2):c.5791C>T (p.Gln1931Ter)-   NM_000059.3(BRCA2):c.6070C>T (p.Gln2024Ter)-   NM_000059.3(BRCA2):c.7024C>T (p.Gln2342Ter)-   NM_000059.3(BRCA2):c.961C>T (p.Gln321Ter)-   NM_000059.3(BRCA2):c.9380G>A (p.Trp3127Ter)-   NM_000059.3(BRCA2):c.8364G>A (p.Trp2788Ter)-   NM_000059.3(BRCA2):c.7758G>A (p.Trp2586Ter)-   NM_000059.3(BRCA2):c.2224C>T (p.Gln742Ter)-   NM_000059.3(BRCA2):c.5101C>T (p.Gln1701Ter)-   NM_000059.3(BRCA2):c.5959C>T (p.Gln1987Ter)-   NM_000059.3(BRCA2):c.7060C>T (p.Gln2354Ter)-   NM_000059.3(BRCA2):c.9100C>T (p.Gln3034Ter)-   NM_000059.3(BRCA2):c.9148C>T (p.Gln3050Ter)-   NM_000059.3(BRCA2):c.9883C>T (p.Gln3295Ter)-   NM_000059.3(BRCA2):c.1414C>T (p.Gln472Ter)-   NM_000059.3(BRCA2):c.1689G>A (p.Trp563Ter)-   NM_000059.3(BRCA2):c.581G>A (p.Trp194Ter)-   NM_000059.3(BRCA2):c.6490C>T (p.Gln2164Ter)-   NM_000059.3(BRCA2):c.7856G>A (p.Trp2619Ter)-   NM_000059.3(BRCA2):c.8970G>A (p.Trp2990Ter)-   NM_000059.3(BRCA2):c.92G>A (p.Trp31Ter)-   NM_000059.3(BRCA2):c.9376C>T (p.Gln3126Ter)-   NM_000059.3(BRCA2):c.93G>A (p.Trp31Ter)-   NM_000059.3(BRCA2):c.1189C>T (p.Gln397Ter)-   NM_000059.3(BRCA2):c.2818C>T (p.Gln940Ter)-   NM_000059.3(BRCA2):c.2979G>A (p.Trp993Ter)-   NM_000059.3(BRCA2):c.3166C>T (p.Gln1056Ter)-   NM_000059.3(BRCA2):c.4285C>T (p.Gln1429Ter)-   NM_000059.3(BRCA2):c.6025C>T (p.Gln2009Ter)-   NM_000059.3(BRCA2):c.772C>T (p.Gln258Ter)-   NM_000059.3(BRCA2):c.7877G>A (p.Trp2626Ter)-   NM_000059.3(BRCA2):c.3109C>T (p.Gln1037Ter)-   NM_000059.3(BRCA2):c.4222C>T (p.Gln1408Ter)-   NM_000059.3(BRCA2):c.7480C>T (p.Arg2494Ter)-   NM_000059.3(BRCA2):c.7878G>A (p.Trp2626Ter)-   NM_000059.3(BRCA2):c.9076C>T (p.Gln3026Ter)-   NM_000059.3(BRCA2):c.1855C>T (p.Gln619Ter)-   NM_000059.3(BRCA2):c.4111C>T (p.Gln1371Ter)-   NM_000059.3(BRCA2):c.5656C>T (p.Gln1886Ter)-   NM_000059.3(BRCA2):c.7757G>A (p.Trp2586Ter)-   NM_000059.3(BRCA2):c.8243G>A (p.Gly2748Asp)-   NM_000059.3(BRCA2):c.8878C>T (p.Gln2960Ter)-   NM_000059.3(BRCA2):c.8487+1G>A-   NM_000059.3(BRCA2):c.8677C>T (p.Gln2893Ter)-   NM_000059.3(BRCA2):c.250C>T (p.Gln84Ter)-   NM_000059.3(BRCA2):c.6124C>T (p.Gln2042Ter)-   NM_000059.3(BRCA2):c.7617+1G>A-   NM_000059.3(BRCA2):c.8575C>T (p.Gln2859Ter)-   NM_000059.3(BRCA2):c.8174G>A (p.Trp2725Ter)-   NM_000059.3(BRCA2):c.3187C>T (p.Gln1063Ter)-   NM_000059.3(BRCA2):c.9381G>A (p.Trp3127Ter)-   NM_000059.3(BRCA2):c.2095C>T (p.Gln699Ter)-   NM_000059.3(BRCA2):c.1642C>T (p.Gln548Ter)-   NM_000059.3(BRCA2):c.8608C>T (p.Gln2870Ter)-   NM_000059.3(BRCA2):c.3412C>T (p.Gln1138Ter)-   NM_000059.3(BRCA2):c.4246C>T (p.Gln1416Ter)-   NM_000059.3(BRCA2):c.6475C>T (p.Gln2159Ter)-   NM_000059.3(BRCA2):c.7366C>T (p.Gln2456Ter)-   NM_000059.3(BRCA2):c.7516C>T (p.Gln2506Ter)-   NM_000059.3(BRCA2):c.8969G>A (p.Trp2990Ter)-   NM_000059.3(BRCA2):c.6487C>T (p.Gln2163Ter)-   NM_000059.3(BRCA2):c.2978G>A (p.Trp993Ter)-   NM_000059.3(BRCA2):c.7615C>T (p.Gln2539Ter)-   NM_000059.3(BRCA2):c.9106C>T (p.Gln3036Ter)    See Table A. Accordingly, an aspect of the invention relates to a    method for treating or preventing Breast-Ovarian Cancer by    correcting one or more pathogenic G-to-A or C-to-T mutations/SNPs,    particularly one or more pathogenic G-to-A or C-to-T mutations/SNPs    present in the BRCA1or BRCA2 gene, and more particularly one or more    pathogenic G-to-A or C-to-T mutations/SNPs described above.

Lynch Syndrome

In some embodiments, the methods, systems, and compositions describedherein are used to correct one or more pathogenic G-to-A or C-to-Tmutations/SNPs associated with Lynch syndrome. In some embodiment, thepathogenic mutations/SNPs are present in at least one gene selected fromMSH6, MSH2, EPCAM, PMS2, and MLH1, including at least the followings:

-   NM_000179.2(MSH6):c.1045C>T (p.Gln349Ter)-   NM_000251.2(MSH2):c.1384C>T (p.Gln462Ter)-   NM_002354.2(EPCAM):c.133C>T (p.Gln45Ter)-   NM_002354.2(EPCAM):c.429G>A (p.Trp143Ter)-   NM_002354.2(EPCAM):c.523C>T (p.Gln175Ter)-   NM_000179.2(MSH6):c.2680C>T (p.Gln894Ter)-   NM_000251.2(MSH2):c.350G>A (p.Trp117Ter)-   NM_000179.2(MSH6):c.2735G>A (p.Trp912Ter)-   NM_000179.2(MSH6):c.3556+1G>A-   NM_000251.2(MSH2):c.388C>T (p.Gln130Ter)-   NM_000535.6(PMS2):c.1912C>T (p.Gln638Ter)-   NM_000535.6(PMS2):c.1891C>T (p.Gln631Ter)-   NM_000249.3(MLH1):c.454-1G>A-   NM_000251.2(MSH2):c.1030C>T (p.Gln344Ter)-   NM_000179.2(MSH6):c.2330G>A (p.Trp777Ter)-   NM_000179.2(MSH6):c.2191C>T (p.Gln731Ter)-   NM_000179.2(MSH6):c.2764C>T (p.Arg922Ter)-   NM_000179.2(MSH6):c.2815C>T (p.Gln939Ter)-   NM_000179.2(MSH6):c.3020G>A (p.Trp1007Ter)-   NM_000179.2(MSH6):c.3436C>T (p.Gln1146Ter)-   NM_000179.2(MSH6):c.3647-1G>A-   NM_000179.2(MSH6):c.3772C>T (p.Gln1258Ter)-   NM_000179.2(MSH6):c.3838C>T (p.Gln1280Ter)-   NM_000179.2(MSH6):c.706C>T (p.Gln236Ter)-   NM_000179.2(MSH6):c.730C>T (p.Gln244Ter)-   NM_000249.3(MLH1):c.1171C>T (p.Gln391Ter)-   NM_000249.3(MLH1):c.1192C>T (p.Gln398Ter)-   NM_000249.3(MLH1):c.1225C>T (p.Gln409Ter)-   NM_000249.3(MLH1):c.1276C>T (p.Gln426Ter)-   NM_000249.3(MLH1):c.1528C>T (p.Gln510Ter)-   NM_000249.3(MLH1):c.1609C>T (p.Gln537Ter)-   NM_000249.3(MLH1):c.1613G>A (p.Trp538Ter)-   NM_000249.3(MLH1):c.1614G>A (p.Trp538Ter)-   NM_000249.3(MLH1):c.1624C>T (p.Gln542Ter)-   NM_000249.3(MLH1):c.1684C>T (p.Gln562Ter)-   NM_000249.3(MLH1):c.1731+1G>A-   NM_000249.3(MLH1):c.1731+5G>A-   NM_000249.3(MLH1):c.1732-1G>A-   NM_000249.3(MLH1):c.1896G>A (p.Glu632=)-   NM_000249.3(MLH1):c.1989+1G>A-   NM_000249.3(MLH1):c.1990-1G>A-   NM_000249.3(MLH1):c.1998G>A (p.Trp666Ter)-   NM_000249.3(MLH1):c.208-1G>A-   NM_000249.3(MLH1):c.2101C>T (p.Gln701Ter)-   NM_000249.3(MLH1):c.2136G>A (p.Trp712Ter)-   NM_000249.3(MLH1):c.2224C>T (p.Gln742Ter)-   NM_000249.3(MLH1):c.230G>A (p.Cys77Tyr)-   NM_000249.3(MLH1):c.256C>T (p.Gln86Ter)-   NM_000249.3(MLH1):c.436C>T (p.Gln146Ter)-   NM_000249.3(MLH1):c.445C>T (p.Gln149Ter)-   NM_000249.3(MLH1):c.545G>A (p.Arg182Lys)-   NM_000249.3(MLH1):c.731G>A (p.Gly244Asp)-   NM_000249.3(MLH1):c.76C>T (p.Gln26Ter)-   NM_000249.3(MLH1):c.842C>T (p.Ala281Val)-   NM_000249.3(MLH1):c.882C>T (p.Leu294=)-   NM_000249.3(MLH1):c.901C>T (p.Gln301Ter)-   NM_000251.2(MSH2):c.1013G>A (p.Gly338Glu)-   NM_000251.2(MSH2):c.1034G>A (p.Trp345Ter)-   NM_000251.2(MSH2):c.1129C>T (p.Gln377Ter)-   NM_000251.2(MSH2):c.1183C>T (p.Gln395Ter)-   NM_000251.2(MSH2):c.1189C>T (p.Gln397Ter)-   NM_000251.2(MSH2):c.1204C>T (p.Gln402Ter)-   NM_000251.2(MSH2):c.1276+1G>A-   NM_000251.2(MSH2):c.1528C>T (p.Gln510Ter)-   NM_000251.2(MSH2):c.1552C>T (p.Gln518Ter)-   NM_000251.2(MSH2):c.1720C>T (p.Gln574Ter)-   NM_000251.2(MSH2):c.1777C>T (p.Gln593Ter)-   NM_000251.2(MSH2):c.1885C>T (p.Gln629Ter)-   NM_000251.2(MSH2):c.2087C>T (p.Pro696Leu)-   NM_000251.2(MSH2):c.2251G>A (p.Gly751Arg)-   NM_000251.2(MSH2):c.2291G>A (p.Trp764Ter)-   NM_000251.2(MSH2):c.2292G>A (p.Trp764Ter)-   NM_000251.2(MSH2):c.2446C>T (p.Gln816Ter)-   NM_000251.2(MSH2):c.2470C>T (p.Gln824Ter)-   NM_000251.2(MSH2):c.2536C>T (p.Gln846Ter)-   NM_000251.2(MSH2):c.2581C>T (p.Gln861Ter)-   NM_000251.2(MSH2):c.2634G>A (p.Glu878=)-   NM_000251.2(MSH2):c.2635C>T (p.Gln879Ter)-   NM_000251.2(MSH2):c.28C>T (p.Gln10Ter)-   NM_000251.2(MSH2):c.472C>T (p.Gln158Ter)-   NM_000251.2(MSH2):c.478C>T (p.Gln160Ter)-   NM_000251.2(MSH2):c.484G>A (p.Gly162Arg)-   NM_000251.2(MSH2):c.490G>A (p.Gly164Arg)-   NM_000251.2(MSH2):c.547C>T (p.Gln183Ter)-   NM_000251.2(MSH2):c.577C>T (p.Gln193Ter)-   NM_000251.2(MSH2):c.643C>T (p.Gln215Ter)-   NM_000251.2(MSH2):c.645+1G>A-   NM_000251.2(MSH2):c.652C>T (p.Gln218Ter)-   NM_000251.2(MSH2):c.754C>T (p.Gln252Ter)-   NM_000251.2(MSH2):c.792+1G>A-   NM_000251.2(MSH2):c.942G>A (p.Gln314=)-   NM_000535.6(PMS2):c.949C>T (p.Gln317Ter)-   NM_000249.3(MLH1):c.306+1G>A-   NM_000249.3(MLH1):c.62C>T (p.Ala21Val)-   NM_000251.2(MSH2):c.1865C>T (p.Pro622Leu)-   NM_000179.2(MSH6):c.426G>A (p.Trp142Ter)-   NM_000251.2(MSH2):c.715C>T (p.Gln239Ter)-   NM_000249.3(MLH1):c.350C>T (p.Thr117Met)-   NM_000251.2(MSH2):c.1915C>T (p.His639Tyr)-   NM_000251.2(MSH2):c.289C>T (p.Gln97Ter)-   NM_000251.2(MSH2):c.2785C>T (p.Arg929Ter)-   NM_000249.3(MLH1):c.131C>T (p.Ser44Phe)-   NM_000249.3(MLH1):c.1219C>T (p.Gln407Ter)-   NM_000249.3(MLH1):c.306+5G>A-   NM_000251.2(MSH2):c.1801C>T (p.Gln601Ter)-   NM_000535.6(PMS2):c.1144+1G>A-   NM_000251.2(MSH2):c.1984C>T (p.Gln662Ter)-   NM_000249.3(MLH1):c.381-1G>A-   NM_000535.6(PMS2):c.631C>T (p.Arg211Ter)-   NM_000251.2(MSH2):c.790C>T (p.Gln264Ter)-   NM_000251.2(MSH2):c.366+1G>A-   NM_000249.3(MLH1):c.298C>T (p.Arg100Ter)-   NM_000179.2(MSH6):c.3013C>T (p.Arg1005Ter)-   NM_000179.2(MSH6):c.694C>T (p.Gln232Ter)-   NM_000179.2(MSH6):c.742C>T (p.Arg248Ter)-   NM_000249.3(MLH1):c.1039-1G>A-   NM_000249.3(MLH1):c.142C>T (p.Gln48Ter)-   NM_000249.3(MLH1):c.1790G>A (p.Trp597Ter)-   NM_000249.3(MLH1):c.1961C>T (p.Pro654Leu)-   NM_000249.3(MLH1):c.2103+1G>A-   NM_000249.3(MLH1):c.2135G>A (p.Trp712Ter)-   NM_000249.3(MLH1):c.588+5G>A-   NM_000249.3(MLH1):c.790+1G>A-   NM_000251.2(MSH2):c.1035G>A (p.Trp345Ter)-   NM_000251.2(MSH2):c.1255C>T (p.Gln419Ter)-   NM_000251.2(MSH2):c.1861C>T (p.Arg621Ter)-   NM_000251.2(MSH2):c.226C>T (p.Gln76Ter)-   NM_000251.2(MSH2):c.2653C>T (p.Gln885Ter)-   NM_000251.2(MSH2):c.508C>T (p.Gln170Ter)-   NM_000251.2(MSH2):c.862C>T (p.Gln288Ter)-   NM_000251.2(MSH2):c.892C>T (p.Gln298Ter)-   NM_000251.2(MSH2):c.970C>T (p.Gln324Ter)-   NM_000179.2(MSH6):c.4001G>A (p.Arg1334Gln)-   NM_000251.2(MSH2):c.1662-1G>A-   NM_000535.6(PMS2):c.1882C>T (p.Arg628Ter)-   NM_000535.6(PMS2):c.2174+1G>A-   NM_000535.6(PMS2):c.2404C>T (p.Arg802Ter)-   NM_000179.2(MSH6):c.3991C>T (p.Arg1331Ter)-   NM_000179.2(MSH6):c.2503C>T (p.Gln835Ter)-   NM_000179.2(MSH6):c.718C>T (p.Arg240Ter)-   NM_000249.3(MLH1):c.1038G>A (p.Gln346=)-   NM_000249.3(MLH1):c.245C>T (p.Thr82Ile)-   NM_000249.3(MLH1):c.83C>T (p.Pro28Leu)-   NM_000249.3(MLH1):c.884G>A (p.Ser295Asn)-   NM_000249.3(MLH1):c.982C>T (p.Gln328Ter)-   NM_000251.2(MSH2):c.1046C>T (p.Pro349Leu)-   NM_000251.2(MSH2):c.1120C>T (p.Gln374Ter)-   NM_000251.2(MSH2):c.1285C>T (p.Gln429Ter)-   NM_000251.2(MSH2):c.1477C>T (p.Gln493Ter)-   NM_000251.2(MSH2):c.2152C>T (p.Gln718Ter)-   NM_000535.6(PMS2):c.703C>T (p.Gln235Ter)-   NM_000249.3(MLH1):c.2141G>A (p.Trp714Ter)-   NM_000251.2(MSH2):c.1009C>T (p.Gln337Ter)-   NM_000251.2(MSH2):c.1216C>T (p.Arg406Ter)-   NM_000179.2(MSH6):c.3202C>T (p.Arg1068Ter)-   NM_000251.2(MSH2):c.1165C>T (p.Arg389Ter)-   NM_000249.3(MLH1):c.1943C>T (p.Pro648Leu)-   NM_000249.3(MLH1):c.200G>A (p.Gly67Glu)-   NM_000249.3(MLH1):c.793C>T (p.Arg265Cys)-   NM_000249.3(MLH1):c.2059C>T (p.Arg687Trp)-   NM_000249.3(MLH1):c.677G>A (p.Arg226Gln)-   NM_000249.3(MLH1):c.2041G>A (p.Ala681Thr)-   NM_000249.3(MLH1):c.1942C>T (p.Pro648Ser)-   NM_000249.3(MLH1):c.676C>T (p.Arg226Ter)-   NM_000251.2(MSH2):c.2038C>T (p.Arg680Ter)-   NM_000179.2(MSH6):c.1483C>T (p.Arg495Ter)-   NM_000179.2(MSH6):c.2194C>T (p.Arg732Ter)-   NM_000179.2(MSH6):c.3103C>T (p.Arg1035Ter)-   NM_000179.2(MSH6):c.892C>T (p.Arg298Ter)-   NM_000249.3(MLH1):c.1459C>T (p.Arg487Ter)-   NM_000249.3(MLH1):c.1731G>A (p.Ser577=)-   NM_000249.3(MLH1):c.184C>T (p.Gln62Ter)-   NM_000249.3(MLH1):c.1975C>T (p.Arg659Ter)-   NM_000249.3(MLH1):c.199G>A (p.Gly67Arg)-   NM_000251.2(MSH2):c.1076+1G>A-   NM_000251.2(MSH2):c.1147C>T (p.Arg383Ter)-   NM_000251.2(MSH2):c.181C>T (p.Gln61Ter)-   NM_000251.2(MSH2):c.212-1G>A-   NM_000251.2(MSH2):c.2131C>T (p.Arg711Ter)-   NM_000535.6(PMS2):c.697C>T (p.Gln233Ter)-   NM_000535.6(PMS2):c.1261C>T (p.Arg421Ter)-   NM_000251.2(MSH2):c.2047G>A (p.Gly683Arg)-   NM_000535.6(PMS2):c.400C>T (p.Arg134Ter)-   NM_000535.6(PMS2):c.1927C>T (p.Gln643Ter)-   NM_000179.2(MSH6):c.1444C>T (p.Arg482Ter)-   NM_000179.2(MSH6):c.2731C>T (p.Arg911Ter)-   NM_000535.6(PMS2):c.943C>T (p.Arg315Ter)    See Table A. Accordingly, an aspect of the invention relates to a    method for treating or preventing Lynch syndrome by correcting one    or more pathogenic G-to-A or C-to-T mutations/SNPs, particularly one    or more pathogenic G-to-A or C-to-T mutations/SNPs present in at    least one gene selected from BCKDHA, BCKDHB, DBT, and DLD, and more    particularly one or more pathogenic G-to-A or C-to-T mutations/SNPs    described above.

Other Genetic Diseases

Pathogenic G-to-A or C-to-T mutations/SNPs associated with additionalgenetic diseases are also reported in the ClinVar database and disclosedin Table A, including but not limited to Marfan syndrome, Hurlersyndrome, Glycogen Storage Disease, and Cystic Fibrosis. Accordingly, anaspect of the invention relates to a method for correcting one or morepathogenic G-to-A or C-to-T mutations/SNPs associated with any of thesediseases, as discussed below.

Marfan Syndrome

In some embodiments, the methods, systems, and compositions describedherein are used to correct one or more pathogenic G-to-A or C-to-Tmutations/SNPs associated with Marfan syndrome. In some embodiment, thepathogenic mutations/SNPs are present in at least the FBN1 gene,including at least the followings:

-   NM_000138.4(FBN1):c.1879C>T (p.Arg627Cys)-   NM_000138.4(FBN1):c.1051C>T (p.Gln351Ter)-   NM_000138.4(FBN1):c.184C>T (p.Arg62Cys)-   NM_000138.4(FBN1):c.2855-1G>A-   NM_000138.4(FBN1):c.3164G>A (p.Cys1055Tyr)-   NM_000138.4(FBN1):c.368G>A (p.Cys123Tyr)-   NM_000138.4(FBN1):c.4955G>A (p.Cys1652Tyr)-   NM_000138.4(FBN1):c.7180C>T (p.Arg2394Ter)-   NM_000138.4(FBN1):c.8267G>A (p.Trp2756Ter)-   NM_000138.4(FBN1):c.1496G>A (p.Cys499Tyr)-   NM_000138.4(FBN1):c.6886C>T (p.Gln2296Ter)-   NM_000138.4(FBN1):c.3373C>T (p.Arg1125Ter)-   NM_000138.4(FBN1):c.640G>A (p.Gly214Ser)-   NM_000138.4(FBN1):c.5038C>T (p.Gln1680Ter)-   NM_000138.4(FBN1):c.434G>A (p.Cys145Tyr)-   NM_000138.4(FBN1):c.2563C>T (p.Gln855Ter)-   NM_000138.4(FBN1):c.7466G>A (p.Cys2489Tyr)-   NM_000138.4(FBN1):c.2089C>T (p.Gln697Ter)-   NM_000138.4(FBN1):c.592C>T (p.Gln198Ter)-   NM_000138.4(FBN1):c.6695G>A (p.Cys2232Tyr)-   NM_000138.4(FBN1):c.6164-1G>A-   NM_000138.4(FBN1):c.5627G>A (p.Cys1876Tyr)-   NM_000138.4(FBN1):c.4061G>A (p.Trp1354Ter)-   NM_000138.4(FBN1):c.1982G>A (p.Cys661Tyr)-   NM_000138.4(FBN1):c.6784C>T (p.Gln2262Ter)-   NM_000138.4(FBN1):c.409C>T (p.Gln137Ter)-   NM_000138.4(FBN1):c.364C>T (p.Arg122Cys)-   NM_000138.4(FBN1):c.3217G>A (p.Glu1073Lys)-   NM_000138.4(FBN1):c.4460-8G>A-   NM_000138.4(FBN1):c.4786C>T (p.Arg1596Ter)-   NM_000138.4(FBN1):c.7806G>A (p.Trp2602Ter)-   NM_000138.4(FBN1):c.247+1G>A-   NM_000138.4(FBN1):c.2495G>A (p.Cys832Tyr)-   NM_000138.4(FBN1):c.493C>T (p.Arg165Ter)-   NM_000138.4(FBN1):c.5504G>A (p.Cys1835Tyr)-   NM_000138.4(FBN1):c.5863C>T (p.Gln1955Ter)-   NM_000138.4(FBN1):c.6658C>T (p.Arg2220Ter)-   NM_000138.4(FBN1):c.7606G>A (p.Gly2536Arg)-   NM_000138.4(FBN1):c.7955G>A (p.Cys2652Tyr)-   NM_000138.4(FBN1):c.3037G>A (p.Gly1013Arg)-   NM_000138.4(FBN1):c.8080C>T (p.Arg2694Ter)-   NM_000138.4(FBN1):c.1633C>T (p.Arg545Cys)-   NM_000138.4(FBN1):c.7205-1G>A-   NM_000138.4(FBN1):c.4621C>T (p.Arg1541Ter)-   NM_000138.4(FBN1):c.1090C>T (p.Arg364Ter)-   NM_000138.4(FBN1):c.1585C>T (p.Arg529Ter)-   NM_000138.4(FBN1):c.4781G>A (p.Gly1594Asp)-   NM_000138.4(FBN1):c.643C>T (p.Arg215Ter)-   NM_000138.4(FBN1):c.3668G>A (p.Cys1223Tyr)-   NM_000138.4(FBN1):c.8326C>T (p.Arg2776Ter)-   NM_000138.4(FBN1):c.6354C>T (p.Ile2118=)-   NM_000138.4(FBN1):c.1468+5G>A-   NM_000138.4(FBN1):c.1546C>T (p.Arg516Ter)-   NM_000138.4(FBN1):c.4615C>T (p.Arg1539Ter)-   NM_000138.4(FBN1):c.5368C>T (p.Arg1790Ter)-   NM_000138.4(FBN1):c.1285C>T (p.Arg429Ter)    See Table A. Accordingly, an aspect of the invention relates to a    method for treating or preventing Marfan syndrome by correcting one    or more pathogenic G-to-A or C-to-T mutations/SNPs, particularly one    or more pathogenic G-to-A or C-to-T mutations/SNPs present in the    FBN1 gene, and more particularly one or more pathogenic G-to-A or    C-to-T mutations/SNPs described above.

Hurler Syndrome

In some embodiments, the methods, systems, and compositions describedherein are used to correct one or more pathogenic G-to-A or C-to-Tmutations/SNPs associated with Hurler syndrome. In some embodiment, thepathogenic mutations/SNPs are present in at least the IDUA gene,including at least the followings:

-   NM_000203.4(IDUA):c.972+1G>A-   NM_000203.4(IDUA):c.1855C>T (p.Arg619Ter)-   NM_000203.4(IDUA):c.152G>A (p.Gly51Asp)-   NM_000203.4(IDUA):c.1205G>A (p.Trp402Ter)-   NM_000203.4(IDUA):c.208C>T (p.Gln70Ter)-   NM_000203.4(IDUA):c.1045G>A (p.Asp349Asn)-   NM_000203.4(IDUA):c.1650+5G>A    See Table A. Accordingly, an aspect of the invention relates to a    method for treating or preventing Hurler syndrome by correcting one    or more pathogenic G-to-A or C-to-T mutations/SNPs, particularly one    or more pathogenic G-to-A or C-to-T mutations/SNPs present in the    IDUA gene, and more particularly one or more pathogenic G-to-A or    C-to-T mutations/SNPs described above.

Glycogen Storage Disease

In some embodiments, the methods, systems, and compositions describedherein are used to correct one or more pathogenic G-to-A or C-to-Tmutations/SNPs associated with Glycogen Storage Disease. In someembodiment, the pathogenic mutations/SNPs are present in at least onegene selected from GAA, AGL, PHKB, PRKAG2, G6PC, PGAM2, GBE1, PYGM, andPFKM, including at least the followings:

-   NM_000152.4(GAA):c.1927G>A (p.Gly643Arg)-   NM_000152.4(GAA):c.2173C>T (p.Arg725Trp)-   NM_000642.2(AGL):c.3980G>A (p.Trp1327Ter)-   NM_000642.2(AGL):c.16C>T (p.Gln6Ter)-   NM_000642.2(AGL):c.2039G>A (p.Trp680Ter)-   NM_000293.2(PHKB):c.1546C>T (p.Gln516Ter)-   NM_016203.3(PRKAG2):c.1592G>A (p.Arg531Gln)-   NM_000151.3(G6PC):c.248G>A (p.Arg83His)-   NM_000151.3(G6PC):c.724C>T (p.Gln242Ter)-   NM_000151.3(G6PC):c.883C>T (p.Arg295Cys)-   NM_000151.3(G6PC):c.247C>T (p.Arg83Cys)-   NM_000151.3(G6PC):c.1039C>T (p.Gln347Ter)-   NM_000152.4(GAA):c.1561G>A (p.Glu521Lys)-   NM_000642.2(AGL):c.2590C>T (p.Arg864Ter)-   NM_000642.2(AGL):c.3682C>T (p.Arg1228Ter)-   NM_000642.2(AGL):c.118C>T (p.Gln40Ter)-   NM_000642.2(AGL):c.256C>T (p.Gln86Ter)-   NM_000642.2(AGL):c.2681+1G>A-   NM_000642.2(AGL):c.2158-1G>A-   NM_000290.3(PGAM2):c.233G>A (p.Trp78Ter)-   NM_000152.4(GAA):c.1548G>A (p.Trp516Ter)-   NM_000152.4(GAA):c.2014C>T (p.Arg672Trp)-   NM_000152.4(GAA):c.546G>A (p.Thr182=)-   NM_000152.4(GAA):c.1802C>T (p.Ser601Leu)-   NM_000152.4(GAA):c.1754+1G>A-   NM_000152.4(GAA):c.1082C>T (p.Pro361Leu)-   NM_000152.4(GAA):c.2560C>T (p.Arg854Ter)-   NM_000152.4(GAA):c.655G>A (p.Gly219Arg)-   NM_000152.4(GAA):c.1933G>A (p.Asp645Asn)-   NM_000152.4(GAA):c.1979G>A (p.Arg660His)-   NM_000152.4(GAA):c.1465G>A (p.Asp489Asn)-   NM_000152.4(GAA):c.2512C>T (p.Gln838Ter)-   NM_000158.3(GBE1):c.1543C>T (p.Arg515Cys)-   NM_005609.3(PYGM):c.1726C>T (p.Arg576Ter)-   NM_005609.3(PYGM):c.1827G>A (p.Lys609=)-   NM_005609.3(PYGM):c.148C>T (p.Arg50Ter)-   NM_005609.3(PYGM):c.613G>A (p.Gly205Ser)-   NM_005609.3(PYGM):c.1366G>A (p.Val456Met)-   NM_005609.3(PYGM):c.1768+1G>A-   NM_001166686.1(PFKM):c.450+1G>A    See Table A. Accordingly, an aspect of the invention relates to a    method for treating or preventing Glycogen Storage Disease by    correcting one or more pathogenic G-to-A or C-to-T mutations/SNPs,    particularly one or more pathogenic G-to-A or C-to-T mutations/SNPs    present in at least one gene selected from GAA, AGL, PHKB, PRKAG2,    G6PC, PGAM2, GBE1, PYGM, and PFKM, and more particularly one or more    pathogenic G-to-A or C-to-T mutations/SNPs described above.

Cystic Fibrosis

In some embodiments, the methods, systems, and compositions describedherein are used to correct one or more pathogenic G-to-A or C-to-Tmutations/SNPs associated with Cystic Fibrosis. In some embodiment, thepathogenic mutations/SNPs are present in the CFTR gene, including atleast the followings:

-   NM_000492.3(CFTR):c.3712C>T (p.Gln1238Ter)-   NM_000492.3(CFTR):c.3484C>T (p.Arg1162Ter)-   NM_000492.3(CFTR):c.1766+1G>A-   NM_000492.3(CFTR):c.1477C>T (p.Gln493Ter)-   NM_000492.3(CFTR):c.2538G>A (p.Trp846Ter)-   NM_000492.3(CFTR):c.2551C>T (p.Arg851Ter)-   NM_000492.3(CFTR):c.3472C>T (p.Arg1158Ter)-   NM_000492.3(CFTR):c.1475C>T (p.Ser492Phe)-   NM_000492.3(CFTR):c.1679G>A (p.Arg560Lys)-   NM_000492.3(CFTR):c.3197G>A (p.Arg1066His)-   NM_000492.3(CFTR):c.3873+1G>A-   NM_000492.3(CFTR):c.3196C>T (p.Arg1066Cys)-   NM_000492.3(CFTR):c.2490+1G>A-   NM_000492.3(CFTR):c.3718-1G>A-   NM_000492.3(CFTR):c.171G>A (p.Trp57Ter)-   NM_000492.3(CFTR):c.3937C>T (p.Gln1313Ter)-   NM_000492.3(CFTR):c.274G>A (p.Glu92Lys)-   NM_000492.3(CFTR):c.1013C>T (p.Thr338Ile)-   NM_000492.3(CFTR):c.3266G>A (p.Trp1089Ter)-   NM_000492.3(CFTR):c.1055G>A (p.Arg352Gln)-   NM_000492.3(CFTR):c.1654C>T (p.Gln552Ter)-   NM_000492.3(CFTR):c.2668C>T (p.Gln890Ter)-   NM_000492.3(CFTR):c.3611G>A (p.Trp1204Ter)-   NM_000492.3(CFTR):c.1585-8G>A-   NM_000492.3(CFTR):c.223C>T (p.Arg75Ter)-   NM_000492.3(CFTR):c.1680-1G>A-   NM_000492.3(CFTR):c.349C>T (p.Arg117Cys)-   NM_000492.3(CFTR):c.1203G>A (p.Trp401Ter)-   NM_000492.3(CFTR):c.1240C>T (p.Gln414Ter)-   NM_000492.3(CFTR):c.1202G>A (p.Trp401Ter)-   NM_000492.3(CFTR):c.1209+1G>A-   NM_000492.3(CFTR):c.115C>T (p.Gln39Ter)-   NM_000492.3(CFTR):c.1116+1G>A-   NM_000492.3(CFTR):c.1393-1G>A-   NM_000492.3(CFTR):c.1573C>T (p.Gln525Ter)-   NM_000492.3(CFTR):c.164+1G>A-   NM_000492.3(CFTR):c.166G>A (p.Glu56Lys)-   NM_000492.3(CFTR):c.170G>A (p.Trp57Ter)-   NM_000492.3(CFTR):c.2053C>T (p.Gln685Ter)-   NM_000492.3(CFTR):c.2125C>T (p.Arg709Ter)-   NM_000492.3(CFTR):c.2290C>T (p.Arg764Ter)-   NM_000492.3(CFTR):c.2353C>T (p.Arg785Ter)-   NM_000492.3(CFTR):c.2374C>T (p.Arg792Ter)-   NM_000492.3(CFTR):c.2537G>A (p.Trp846Ter)-   NM_000492.3(CFTR):c.292C>T (p.Gln98Ter)-   NM_000492.3(CFTR):c.2989-1G>A-   NM_000492.3(CFTR):c.3293G>A (p.Trp1098Ter)-   NM_000492.3(CFTR):c.4144C>T (p.Gln1382Ter)-   NM_000492.3(CFTR):c.4231C>T (p.Gln1411Ter)-   NM_000492.3(CFTR):c.4234C>T (p.Gln1412Ter)-   NM_000492.3(CFTR):c.579+5G>A-   NM_000492.3(CFTR):c.595C>T (p.His199Tyr)-   NM_000492.3(CFTR):c.613C>T (p.Pro205Ser)-   NM_000492.3(CFTR):c.658C>T (p.Gln220Ter)-   NM_000492.3(CFTR):c.1117-1G>A-   NM_000492.3(CFTR):c.3294G>A (p.Trp1098Ter)-   NM_000492.3(CFTR):c.1865G>A (p.Gly622Asp)-   NM_000492.3(CFTR):c.743+1G>A-   NM_000492.3(CFTR):c.1679+1G>A-   NM_000492.3(CFTR):c.1657C>T (p.Arg553Ter)-   NM_000492.3(CFTR):c.1675G>A (p.Ala559Thr)-   NM_000492.3(CFTR):c.165-1G>A-   NM_000492.3(CFTR):c.200C>T (p.Pro67Leu)-   NM_000492.3(CFTR):c.2834C>T (p.Ser945Leu)-   NM_000492.3(CFTR):c.3846G>A (p.Trp1282Ter)-   NM_000492.3(CFTR):c.1652G>A (p.Gly551Asp)-   NM_000492.3(CFTR):c.4426C>T (p.Gln1476Ter)-   NM_000492.3:c. 3718-2477C>T-   NM_000492.3(CFTR):c.2988+1G>A-   NM_000492.3(CFTR):c.2657+5G>A-   NM_000492.3(CFTR):c.2988G>A (p.Gln996=)-   NM_000492.3(CFTR):c.274-1G>A-   NM_000492.3(CFTR):c.3612G>A (p.Trp1204Ter)-   NM_000492.3(CFTR):c.1646G>A (p.Ser549Asn)-   NM_000492.3(CFTR):c.3752G>A (p.Ser1251Asn)-   NM_000492.3(CFTR):c.4046G>A (p.Gly1349Asp)-   NM_000492.3(CFTR):c.532G>A (p.Gly178Arg)-   NM_000492.3(CFTR):c.3731G>A (p.Gly1244Glu)-   NM_000492.3(CFTR):c.1651G>A (p.Gly551Ser)-   NM_000492.3(CFTR):c.1585-1G>A-   NM_000492.3(CFTR):c.1000C>T (p.Arg334Trp)-   NM_000492.3(CFTR):c.254G>A (p.Gly85Glu)-   NM_000492.3(CFTR):c.1040G>A (p.Arg347His)-   NM_000492.3(CFTR):c.273+1G>A    See Table A. Accordingly, an aspect of the invention relates to a    method for treating or preventing Cystic Fibrosis by correcting one    or more pathogenic G-to-A or C-to-T mutations/SNPs, particularly one    or more pathogenic G-to-A or C-to-T mutations/SNPs present in the    CFTR gene, and more particularly one or more pathogenic G-to-A or    C-to-T mutations/SNPs described above.

In some embodiments, the methods, systems, and compositions describedherein are used to correct a pathogenic A-to-G (A>G) mutation or SNPbelieved to be associated with familial 2 breast-ovarian cancer, whereinthe pathogenic A>G mutation or SNP is located in the BRCA2 gene (HGVS:U43746.1:n.7829+1G>A). Accordingly, an additional aspect of theinvention relates to a method for treating or preventing familial 2breast-ovarian cancer by correcting the aforementioned pathogenic A>Gmutation or SNP.

In some embodiments, the methods, systems, and compositions describedherein are used to correct a pathogenic A-to-G (A>G) mutation or SNPbelieved to be associated with hereditary factor IX deficiency, whereinthe pathogenic A>G mutation or SNP is located at GRCh38: ChrX: 139537145in the F9 gene, which results in an Arg to Gln substitution.Accordingly, an additional aspect of the invention relates to a methodfor treating or preventing hereditary factor IX deficiency by correctingthe aforementioned pathogenic A>G mutation or SNP.

In some embodiments, the methods, systems, and compositions describedherein are used to correct a pathogenic A-to-G (A>G) mutation or SNPbelieved to be associated with beta-plus-thalassemia, beta thalassemia,and beta thalassemia major, wherein the pathogenic A>G mutation or SNPis located at GRCh38: Chr11: 5226820 in the HBB gene. Accordingly, anadditional aspect of the invention relates to a method for treating orpreventing with beta-plus-thalassemia, beta thalassemia, and betathalassemia major by correcting the aforementioned pathogenic A>Gmutation or SNP.

In some embodiments, the methods, systems, and compositions describedherein are used to correct a pathogenic A-to-G (A>G) mutation or SNPbelieved to be associated with Marfan syndrome, wherein the pathogenicA>G mutation or SNP is located in the FBN1 gene (IVS2DS, G-A, +1), asreported by Yamamoto et al. J Hum Genet. 2000; 45(2):115-8. Accordingly,an additional aspect of the invention relates to a method for treatingor preventing Marfan syndrome by correcting the aforementionedpathogenic A>G mutation or SNP.

In some embodiments, the methods, systems, and compositions describedherein are used to correct a pathogenic A-to-G (A>G) mutation or SNPbelieved to be associated with Wiskott-Aldrich syndrome, wherein thepathogenic A>G mutation or SNP is located at position −1 of intro 6 ofthe WAS gene (IVS6AS, G-A, −1), as reported by Kwan et al. (1995).Accordingly, an additional aspect of the invention relates to a methodfor treating or preventing Wiskott-Aldrich syndrome by correcting theaforementioned pathogenic A>G mutation or SNP.

In some embodiments, the methods, systems, and compositions describedherein are used to correct a pathogenic A-to-G (A>G) mutation or SNPbelieved to be associated with cystic fibrosis, wherein the pathogenicA>G mutation or SNP is located at GRCh38: Chr7:117590440 in the CFTRgene. Accordingly, an additional aspect of the invention relates to amethod for treating or preventing cystic fibrosis by correcting theaforementioned pathogenic A>G mutation or SNP.

In some embodiments, the methods, systems, and compositions describedherein are used to correct a pathogenic A-to-G (A>G) mutation or SNPbelieved to be associated with cystic fibrosis and hereditarypancreatitis, wherein the pathogenic A>G mutation or SNP is locatedGRCh38: Chr7:117606754 in the CFTR gene. Accordingly, an additionalaspect of the invention relates to a method for treating or preventingcystic fibrosis and hereditary pancreatitis by correcting theaforementioned pathogenic A>G mutation or SNP.

In some embodiments, the methods, systems, and compositions describedherein are used to correct a pathogenic A-to-G (A>G) mutation or SNPbelieved to be associated with cystic fibrosis, wherein the pathogenicA>G mutation or SNP is located at GRCh38: Chr7: 117587738 in the CFTRgene. Accordingly, an additional aspect of the invention relates to amethod for treating or preventing cystic fibrosis by correcting theaforementioned pathogenic A>G mutation or SNP.

In some embodiments, the methods, systems, and compositions describedherein are used to correct a pathogenic A-to-G (A>G) mutation or SNPbelieved to be associated with Turcot syndrome and Lynch syndrome,wherein the pathogenic A>G mutation or SNP is located at GRCh38:Chr2:47470964 in the MSH2 gene. Accordingly, an additional aspect of theinvention relates to a method for treating or preventing Turcot syndromeand Lynch syndrome by correcting the aforementioned pathogenic A>Gmutation or SNP.

In some embodiments, the methods, systems, and compositions describedherein are used to correct a pathogenic A-to-G (A>G) mutation or SNPbelieved to be associated with cystic fibrosis, wherein the pathogenicA>G mutation or SNP is located at GRCh38: Chr7: 117642437 in the CFTRgene. Accordingly, an additional aspect of the invention relates to amethod for treating or preventing cystic fibrosis by correcting theaforementioned pathogenic A>G mutation or SNP.

In some embodiments, the methods, systems, and compositions describedherein are used to correct a pathogenic A-to-G (A>G) mutation or SNPbelieved to be associated with Lynch syndrome II and Lynch syndrome,wherein the pathogenic A>G mutation or SNP is located at GRCh38:Chr3:37001058 in the MLH1 gene. Accordingly, an additional aspect of theinvention relates to a method for treating or preventing Lynch syndromeII and Lynch syndrome by correcting the aforementioned pathogenic A>Gmutation or SNP.

In some embodiments, the methods, systems, and compositions describedherein are used to correct a pathogenic A-to-G (A>G) mutation or SNPbelieved to be associated with cystic fibrosis, wherein the pathogenicA>G mutation or SNP is located at GRCh38: Chr7: 117642594 in the CFTRgene. Accordingly, an additional aspect of the invention relates to amethod for treating or preventing cystic fibrosis by correcting theaforementioned pathogenic A>G mutation or SNP.

In some embodiments, the methods, systems, and compositions describedherein are used to correct a pathogenic A-to-G (A>G) mutation or SNPbelieved to be associated with cystic fibrosis, wherein the pathogenicA>G mutation or SNP is located at GRCh38: Chr7: 117592658 in the CFTRgene. Accordingly, an additional aspect of the invention relates to amethod for treating or preventing cystic fibrosis by correcting theaforementioned pathogenic A>G mutation or SNP.

In some embodiments, the methods, systems, and compositions describedherein are used to correct a pathogenic A-to-G (A>G) mutation or SNPbelieved to be associated with familial 1 breast-ovarian cancer,hereditary breast and ovarian cancer syndrome, and hereditarycancer-predisposing syndrome, wherein the pathogenic A>G mutation or SNPis located at GRCh38: Chr17:43057051 in the BRCA1 gene. Accordingly, anadditional aspect of the invention relates to a method for treating orpreventing familial 1 breast-ovarian cancer, hereditary breast andovarian cancer syndrome, and hereditary cancer-predisposing syndrome bycorrecting the aforementioned pathogenic A>G mutation or SNP.

In some embodiments, the methods, systems, and compositions describedherein are used to correct a pathogenic A-to-G (A>G) mutation or SNPbelieved to be associated with dihydropyrimidine dehydrogenasedeficiency, Hirschsprung disease 1, fluorouracil response, pyrimidineanalogues response—toxicity/ADR, capecitabine response—toxicity/ADR,fluorouracil response—toxicity/ADR, tegafur response—toxicity/ADR,wherein the pathogenic A>G mutation or SNP is located at GRCh38:Chr1:97450058 in the DPYD gene. Accordingly, an additional aspect of theinvention relates to a method for treating or preventingdihydropyrimidine dehydrogenase deficiency, Hirschsprung disease 1,fluorouracil response, pyrimidine analogues response—toxicity/ADR,capecitabine response—toxicity/ADR, fluorouracil response—toxicity/ADR,tegafur response—toxicity/ADR by correcting the aforementionedpathogenic A>G mutation or SNP.

In some embodiments, the methods, systems, and compositions describedherein are used to correct a pathogenic A-to-G (A>G) mutation or SNPbelieved to be associated with Lynch syndrome, wherein the pathogenicA>G mutation or SNP is located at GRCh38: Chr2:47478520 in the MSH2gene. Accordingly, an additional aspect of the invention relates to amethod for treating or preventing the Lynch syndrome by correcting theaforementioned pathogenic A>G mutation or SNP.

In some embodiments, the methods, systems, and compositions describedherein are used to correct a pathogenic A-to-G (A>G) mutation or SNPbelieved to be associated with Lynch syndrome, wherein the pathogenicA>G mutation or SNP is located at GRCh38: Chr3:37011819 in the MLH1gene. Accordingly, an additional aspect of the invention relates to amethod for treating or preventing the Lynch syndrome by correcting theaforementioned pathogenic A>G mutation or SNP.

In some embodiments, the methods, systems, and compositions describedherein are used to correct a pathogenic A-to-G (A>G) mutation or SNPbelieved to be associated with Lynch syndrome, wherein the pathogenicA>G mutation or SNP is located at GRCh38: Chr3: 37014545 in the MLH1gene. Accordingly, an additional aspect of the invention relates to amethod for treating or preventing the Lynch syndrome by correcting theaforementioned pathogenic A>G mutation or SNP.

In some embodiments, the methods, systems, and compositions describedherein are used to correct a pathogenic A-to-G (A>G) mutation or SNPbelieved to be associated with Lynch syndrome, wherein the pathogenicA>G mutation or SNP is located at GRCh38: Chr3: 37011867 in the MLH1gene. Accordingly, an additional aspect of the invention relates to amethod for treating or preventing the Lynch syndrome by correcting theaforementioned pathogenic A>G mutation or SNP.

In some embodiments, the methods, systems, and compositions describedherein are used to correct a pathogenic A-to-G (A>G) mutation or SNPbelieved to be associated with Lynch syndrome, wherein the pathogenicA>G mutation or SNP is located at GRCh38: Chr3: 37025636 in the MLH1gene. Accordingly, an additional aspect of the invention relates to amethod for treating or preventing the Lynch syndrome by correcting theaforementioned pathogenic A>G mutation or SNP.

In some embodiments, the methods, systems, and compositions describedherein are used to correct a pathogenic A-to-G (A>G) mutation or SNPbelieved to be associated with Lynch syndrome, wherein the pathogenicA>G mutation or SNP is located at GRCh38: Chr3: 37004475 in the MLH1gene. Accordingly, an additional aspect of the invention relates to amethod for treating or preventing the Lynch syndrome by correcting theaforementioned pathogenic A>G mutation or SNP.

In some embodiments, the methods, systems, and compositions describedherein are used to correct a pathogenic A-to-G (A>G) mutation or SNPbelieved to be associated with Lynch syndrome and hereditarycancer-predisposing syndrome, wherein the pathogenic A>G mutation or SNPis located at GRCh38: Chr2:47416430 in the MSH2 gene. Accordingly, anadditional aspect of the invention relates to a method for treating orpreventing Lynch syndrome and hereditary cancer-predisposing syndrome bycorrecting the aforementioned pathogenic A>G mutation or SNP.

In some embodiments, the methods, systems, and compositions describedherein are used to correct a pathogenic A-to-G (A>G) mutation or SNPbelieved to be associated with Lynch syndrome and hereditarycancer-predisposing syndrome, wherein the pathogenic A>G mutation or SNPis located at GRCh38: Chr2: 47408400 in the MSH2 gene. Accordingly, anadditional aspect of the invention relates to a method for treating orpreventing Lynch syndrome and hereditary cancer-predisposing syndrome bycorrecting the aforementioned pathogenic A>G mutation or SNP.

In some embodiments, the methods, systems, and compositions describedherein are used to correct a pathogenic A-to-G (A>G) mutation or SNPbelieved to be associated with Lynch syndrome and hereditarycancer-predisposing syndrome, wherein the pathogenic A>G mutation or SNPis located at GRCh38: Chr3:36996710 in the MLH1 gene. Accordingly, anadditional aspect of the invention relates to a method for treating orpreventing Lynch syndrome and hereditary cancer-predisposing syndrome bycorrecting the aforementioned pathogenic A>G mutation or SNP.

In some embodiments, the methods, systems, and compositions describedherein are used to correct a pathogenic A-to-G (A>G) mutation or SNPbelieved to be associated with familial 1 breast-ovarian cancer, whereinthe pathogenic A>G mutation or SNP is located at GRCh38: Chr17:43067696in the BRCA1 gene. Accordingly, an additional aspect of the inventionrelates to a method for treating or preventing familial 1 breast-ovariancancer by correcting the aforementioned pathogenic A>G mutation or SNP.

In some embodiments, the methods, systems, and compositions describedherein are used to correct a pathogenic A-to-G (A>G) mutation or SNPbelieved to be associated with familial 2 breast-ovarian cancer andhereditary breast and ovarian cancer syndrome, wherein the pathogenicA>G mutation or SNP is located at GRCh38: Chr13:32356610 in the BRCA2gene. Accordingly, an additional aspect of the invention relates to amethod for treating or preventing familial 2 breast-ovarian cancer andhereditary breast and ovarian cancer syndrome by correcting theaforementioned pathogenic A>G mutation or SNP.

In some embodiments, the methods, systems, and compositions describedherein are used to correct a pathogenic A-to-G (A>G) mutation or SNPbelieved to be associated with primary dilated cardiomyopathy andprimary familial hypertrophic cardiomyopathy, wherein the pathogenic A>Gmutation or SNP is located at GRCh38: Chr14:23419993 in the MYH7 gene.Accordingly, an additional aspect of the invention relates to a methodfor treating or preventing primary dilated cardiomyopathy and primaryfamilial hypertrophic cardiomyopathy by correcting the aforementionedpathogenic A>G mutation or SNP.

In some embodiments, the methods, systems, and compositions describedherein are used to correct a pathogenic A-to-G (A>G) mutation or SNPbelieved to be associated with primary familial hypertrophiccardiomyopathy, camptocormism, and hypertrophic cardiomyopathy, whereinthe pathogenic A>G mutation or SNP is located at GRCh38: Chr14:23415225in the MYH7 gene. Accordingly, an additional aspect of the inventionrelates to a method for treating or preventing primary familialhypertrophic cardiomyopathy, camptocormism, and hypertrophiccardiomyopathy by correcting the aforementioned pathogenic A>G mutationor SNP.

In some embodiments, the methods, systems, and compositions describedherein are used to correct a pathogenic A-to-G (A>G) mutation or SNPbelieved to be associated with familial cancer of breast, familial 2breast-ovarian cancer, hereditary breast and ovarian cancer syndrome,and hereditary cancer-predisposing syndrome, wherein the pathogenic A>Gmutation or SNP is located at GRCh38: Chr13:32357741 in the BRCA2 gene.Accordingly, an additional aspect of the invention relates to a methodfor treating or preventing the familial cancer of breast, familial 2breast-ovarian cancer, hereditary breast and ovarian cancer syndrome,and hereditary cancer-predisposing syndrome by correcting theaforementioned pathogenic A>G mutation or SNP.

In some embodiments, the methods, systems, and compositions describedherein are used to correct a pathogenic A-to-G (A>G) mutation or SNPbelieved to be associated with primary dilated cardiomyopathy,hypertrophic cardiomyopathy, cardiomyopathy, and left ventricularnoncompaction, wherein the pathogenic A>G mutation or SNP is located atGRCh38: Chr14:23431584 in the MYH7 gene. Accordingly, an additionalaspect of the invention relates to a method for treating or preventingprimary dilated cardiomyopathy, hypertrophic cardiomyopathy,cardiomyopathy, and left ventricular noncompaction by correcting theaforementioned pathogenic A>G mutation or SNP.

In some embodiments, the methods, systems, and compositions describedherein are used to correct a pathogenic A-to-G (A>G) mutation or SNPbelieved to be associated with familial 1 breast-ovarian cancer,hereditary breast and ovarian cancer syndrome, and hereditarycancer-predisposing syndrome, wherein the pathogenic A>G mutation or SNPis located at GRCh38: Chr17:43067607 in the BRCA1 gene. Accordingly, anadditional aspect of the invention relates to a method for treating orpreventing familial 1 breast-ovarian cancer, hereditary breast andovarian cancer syndrome, and hereditary cancer-predisposing syndrome bycorrecting the aforementioned pathogenic A>G mutation or SNP.

In some embodiments, the methods, systems, and compositions describedherein are used to correct a pathogenic A-to-G (A>G) mutation or SNPbelieved to be associated with familial 1 breast-ovarian cancer,hereditary breast and ovarian cancer syndrome, hereditarycancer-predisposing syndrome, and breast cancer, wherein the pathogenicA>G mutation or SNP is located at GRCh38: Chr17:43047666 in the BRCA1gene. Accordingly, an additional aspect of the invention relates to amethod for treating or preventing familial 1 breast-ovarian cancer,hereditary breast and ovarian cancer syndrome, hereditarycancer-predisposing syndrome, and breast cancer by correcting theaforementioned pathogenic A>G mutation or SNP.

In some embodiments, the methods, systems, and compositions describedherein are used to correct a pathogenic A-to-G (A>G) mutation or SNPbelieved to be associated with familial 2 breast-ovarian cancer,hereditary breast and ovarian cancer syndrome, and hereditarycancer-predisposing syndrome, wherein the pathogenic A>G mutation or SNPis located at GRCh38: Chr13:32370558 in the BRCA2 gene. Accordingly, anadditional aspect of the invention relates to a method for treating orpreventing familial 2 breast-ovarian cancer, hereditary breast andovarian cancer syndrome, and hereditary cancer-predisposing syndrome bycorrecting the aforementioned pathogenic A>G mutation or SNP.

In some embodiments, the methods, systems, and compositions describedherein are used to correct a pathogenic A-to-G (A>G) mutation or SNPbelieved to be associated with familial 1 breast-ovarian cancer,hereditary breast and ovarian cancer syndrome, hereditarycancer-predisposing syndrome, and breast cancer, wherein the pathogenicA>G mutation or SNP is located at GRCh38: Chr17:43074330 in the BRCA1gene. Accordingly, an additional aspect of the invention relates to amethod for treating or preventing familial 1 breast-ovarian cancer,hereditary breast and ovarian cancer syndrome, hereditarycancer-predisposing syndrome, and breast cancer by correcting theaforementioned pathogenic A>G mutation or SNP.

In some embodiments, the methods, systems, and compositions describedherein are used to correct a pathogenic A-to-G (A>G) mutation or SNPbelieved to be associated with familial 1 breast-ovarian cancer,hereditary breast and ovarian cancer syndrome, and hereditarycancer-predisposing syndrome, wherein the pathogenic A>G mutation or SNPis located at GRCh38: Chr17: 43082403 in the BRCA1 gene. Accordingly, anadditional aspect of the invention relates to a method for treating orpreventing familial 1 breast-ovarian cancer, hereditary breast andovarian cancer syndrome, and hereditary cancer-predisposing syndrome bycorrecting the aforementioned pathogenic A>G mutation or SNP.

In some embodiments, the methods, systems, and compositions describedherein are used to correct a pathogenic C-to-T (C>T) mutation or SNPbelieved to be associated with cystic fibrosis and hereditarypancreatitis, wherein the pathogenic C>T mutation or SNP is located atGRCh38: Chr7:117639961 in the CFTR gene. Accordingly, an additionalaspect of the invention relates to a method for treating or preventingthe cystic fibrosis and hereditary pancreatitis by correcting theaforementioned pathogenic C>T mutation or SNP.

In some embodiments, the methods, systems, and compositions describedherein are used to correct a pathogenic C-to-T (C>T) mutation or SNPbelieved to be associated with familial 2 breast-ovarian cancer, whereinthe pathogenic C>T mutation or SNP is located at GRCh38: Chr13:32336492in the BRCA2 gene. Accordingly, an additional aspect of the inventionrelates to a method for treating or preventing the familial 2breast-ovarian cancer by correcting the aforementioned pathogenic C>Tmutation or SNP.

In some embodiments, the methods, systems, and compositions describedherein are used to correct a pathogenic C-to-T (C>T) mutation or SNPbelieved to be associated with familial 1 breast-ovarian cancer, whereinthe pathogenic C>T mutation or SNP is located at GRCh38: Chr17:43063365in the BRCA1 gene. Accordingly, an additional aspect of the inventionrelates to a method for treating or preventing the familial 1breast-ovarian cancer by correcting the aforementioned pathogenic C>Tmutation or SNP.

In some embodiments, the methods, systems, and compositions describedherein are used to correct a pathogenic C-to-T (C>T) mutation or SNPbelieved to be associated with familial 1 breast-ovarian cancer, whereinthe pathogenic C>T mutation or SNP is located at GRCh38: Chr17:43093613in the BRCA1 gene. Accordingly, an additional aspect of the inventionrelates to a method for treating or preventing the familial 1breast-ovarian cancer by correcting the aforementioned pathogenic C>Tmutation or SNP.

In some embodiments, the methods, systems, and compositions describedherein are used to correct a pathogenic C-to-T (C>T) mutation or SNPbelieved to be associated with familial cancer of breast, and familial 1breast-ovarian cancer, wherein the pathogenic C>T mutation or SNP islocated at at GRCh38: Chr17:43093931 of the BRCA1 gene. Accordingly, anadditional aspect of the invention relates to a method for treating orpreventing familial cancer of breast, and familial 1 breast-ovariancancer by correcting the aforementioned pathogenic C>T mutation or SNP.

In some embodiments, the methods, systems, and compositions describedherein are used to correct a pathogenic C-to-T (C>T) mutation or SNPbelieved to be associated with familial hypertrophic cardiomyopathy 1,primary familial hypertrophic cardiomyopathy, and hypertrophiccardiomyopathy, wherein the pathogenic C>T mutation or SNP is located atGRCh38: Chr14:23429279 of the MYH7 gene. Accordingly, an additionalaspect of the invention relates to a method for treating or preventingfamilial hypertrophic cardiomyopathy 1, primary familial hypertrophiccardiomyopathy, and hypertrophic cardiomyopathy by correcting theaforementioned pathogenic C>T mutation or SNP.

In some embodiments, the methods, systems, and compositions describedherein are used to correct a pathogenic C-to-T (C>T) mutation or SNPbelieved to be associated with familial 2 breast-ovarian cancer,hereditary breast and ovarian cancer syndrome, and hereditarycancer-predisposing syndrome, wherein the pathogenic C>T mutation or SNPis located at GRCh38: Chr13:32356472 of the BRCA2 gene. Accordingly, anadditional aspect of the invention relates to a method for treating orpreventing familial 2 breast-ovarian cancer, hereditary breast andovarian cancer syndrome, and hereditary cancer-predisposing syndrome bycorrecting the aforementioned pathogenic C>T mutation or SNP.

In some embodiments, the methods, systems, and compositions describedherein are used to correct a pathogenic C-to-T (C>T) mutation or SNPbelieved to be associated with familial hypertrophic cardiomyopathy 1,primary familial hypertrophic cardiomyopathy, familial restrictivecardiomyopathy, and hypertrophic cardiomyopathy, wherein the pathogenicC>T mutation or SNP is located at GRCh38: Chr14:23429005 in the MYH7gene. Accordingly, an additional aspect of the invention relates to amethod for treating or preventing familial hypertrophic cardiomyopathy1, primary familial hypertrophic cardiomyopathy, familial restrictivecardiomyopathy, and hypertrophic cardiomyopathy by correcting theaforementioned pathogenic C>T mutation or SNP.

Additional pathogenic A>G mutations and SNPs are found in the ClinVardatabase Accordingly, an additional aspect of the present disclosurerelates to correction of a pathogenic A>G mutation or SNP listed inClinVar using the methods, systems, and compositions described herein totreat or prevent a disease or condition associated therewith.

Additional pathogenic C>T mutations and SNPs are also found in theClinVar database. Accordingly, an additional aspect of the presentdisclosure relates to correction of a pathogenic C>T mutation or SNPlisted in ClinVar using the methods, systems, and compositions describedherein to treat or prevent a disease or condition associated therewith.

In one aspect, the invention described herein provides methods formodifying an cytidine residue at a target locus with the aim ofremedying and/or preventing a diseased condition that is or is likely tobe caused by a T(U)-to-C or A-to-G point mutation or a pathogenic singlenucleotide polymorphism (SNP).

Pathogenic T(U)-to-C or A-to-G mutations/SNPs associated with variousdiseases are reported in the ClinVar database, including but not limitedto genetic diseases, cancer, metabolic diseases, or lysosomal storagediseases. Accordingly, an aspect of the invention relates to a methodfor correcting one or more pathogenic T(U)-to-C or A-to-G mutations/SNPsassociated with any of these diseases, as discussed below.

In some embodiments, the methods, systems, and compositions describedherein are used to correct one or more pathogenic T(U)-to-C or A-to-Gmutations/SNPs reported in the ClinVar database. In some embodiments,the methods, systems, and compositions described herein are used tocorrect one or more pathogenic T(U)-to-C or A-to-G mutations/SNPsassociated with any of the diseases or disorders disclosed inWO2017/070632, titled “Nucleobase Editor and Uses Thereof,” which isincorporated herein by reference in its entirety. Exemplary diseases ordisorders that may be treated include, without limitation,3-Methylglutaconic aciduria type 2, 46,XY gonadal dysgenesis,4-Alpha-hydroxyphenylpyruvate hydroxylase deficiency,6-pyruvoyl-tetrahydropterin synthase deficiency, achromatopsia,Acid-labile subunit deficiency, Acrodysostosis, acroerythrokeratoderma,ACTH resistance, ACTH-independent macronodular adrenal hyperplasia,Activated PBK-delta syndrome, Acute intermittent porphyria, Acutemyeloid leukemia, Adams-Oliver syndrome 1/5/6, Adenylosuccinate lyasedeficiency, Adrenoleukodystrophy, Adult neuronal ceroid lipofuscinosis,Adult onset ataxia with oculomotor apraxia, Advanced sleep phasesyndrome, Age-related macular degeneration, Alagille syndrome, Alexanderdisease, Allan-Herndon-Dudley syndrome, Alport syndrome, X-linkedrecessive, Alternating hemiplegia of childhood, Alveolar capillarydysplasia with misalignment of pulmonary veins, Amelogenesis imperfecta,Amyloidogenic transthyretin amyloidosis, Amyotrophic lateral sclerosis,Anemia (nonspherocytic hemolytic, due to G6PD deficiency), Anemia(sideroblastic, pyridoxine-refractory, autosomal recessive), Anonychia,Antithrombin III deficiency, Aortic aneurysm, Aplastic anemia,Apolipoprotein C2 deficiency, Apparent mineralocorticoid excess,Aromatase deficiency, Arrhythmogenic right ventricular cardiomyopathy,Familial hypertrophic cardiomyopathy, Hypertrophic cardiomyopathy,Arthrogryposis multiplex congenital, Aspartylglycosaminuria,Asphyxiating thoracic dystrophy, Ataxia with vitamin E deficiency,Ataxia (spastic), Atrial fibrillation, Atrial septal defect, atypicalhemolytic-uremic syndrome, autosomal dominant CD11C+/CD1C+ dendriticcell deficiency, Autosomal dominant progressive external ophthalmoplegiawith mitochondrial DNA deletions, Baraitser-Winter syndrome, Barttersyndrome, Basa ganglia calcification, Beckwith-Wiedemann syndrome,Benign familial neonatal seizures, Benign scapuloperoneal musculardystrophy, Bernard Soulier syndrome, Beta thalassemia intermedia,Beta-D-mannosidosis, Bietti crystalline corneoretinal dystrophy, Bileacid malabsorption, Biotinidase deficiency, Borjeson-Forssman-Lehmannsyndrome, Boucher Neuhauser syndrome, Bowen-Conradi syndrome,Brachydactyly, Brown-Vialetto-Van laere syndrome, Brugada syndrome,Cardiac arrhythmia, Cardiofaciocutaneous syndrome, Cardiomyopathy,Carnevale syndrome, Carnitine palmitoyltransferase II deficiency,Carpenter syndrome, Cataract, Catecholaminergic polymorphic ventriculartachycardia, Central core disease, Centromeric instability ofchromosomes 1,9 and 16 and immunodeficiency, Cerebral autosomal dominantarteriopathy, Cerebro-oculo-facio-skeletal syndrome, Ceroidlipofuscinosis, Charcot-Marie-Tooth disease, Cholestanol storagedisease, Chondrocalcinosis, Chondrodysplasia, Chronic progressivemultiple sclerosis, Coenzyme Q10 deficiency, Cohen syndrome, Combineddeficiency of factor V and factor VIII, Combined immunodeficiency,Combined oxidative phosphorylation deficiency, Combined partial 17-alpha-hydroxylase/17,20-lyase deficiency, Complement factor ddeficiency, Complete combined 17-alpha-hydroxylase/17,20-lyasedeficiency, Cone-rod dystrophy, Congenital contractural arachnodactyly,Congenital disorder of glycosylation, Congenital lipomatous overgrowth,Neoplasm of ovary, PIK3CA Related Overgrowth Spectrum, Congenital longQT syndrome, Congenital muscular dystrophy, Congenital muscularhypertrophy-cerebral syndrome, Congenital myasthenic syndrome,Congenital myopathy with fiber type di sproportion, Eichsfeld typecongenital muscular dystrophy, Congenital stationary night blindness,Corneal dystrophy, Cornelia de Lange syndrome, Craniometaphysealdysplasia, Crigler Najjar syndrome, Crouzon syndrome, Cutis laxa withosteodystrophy, Cyanosis, Cystic fibrosis, Cystinosis, Cytochrome-coxidase deficiency, Mitochondrial complex I deficiency,D-2-hydroxyglutaric aciduria, Danon disease, Deafness with labyrinthineaplasia microtia and microdontia (LAMM), Deafness, Deficiency ofacetyl-CoA acetyltransferase, Deficiency of ferroxidase, Deficiency ofUDPglucose-hexose-1-phosphate uridylyltransferase, Dejerine-Sottasdisease, Desbuquois syndrome, DFNA, Diabetes mellitus type 2,Diabetes-deafness syndrome, Diamond-Blackfan anemia, Diastrophicdysplasia, Dihydropteridine reductase deficiency, Dihydropyrimidinasedeficiency, Dilated cardiomyopathy, Disseminated atypical mycobacterialinfection, Distal arthrogryposis, Distal hereditary motor neuronopathy,Donnai Barrow syndrome, Duchenne muscular dystrophy, Becker musculardystrophy, Dyschromatosis universalis hereditaria, Dyskeratosiscongenital, Dystonia, Early infantile epileptic encephalopathy,Ehlers-Danlos syndrome, Eichsfeld type congenital muscular dystrophy,Emery-Dreifuss muscular dystrophy, Enamel-renal syndrome, Epidermolysisbullosa dystrophica inversa, Epidermolysis bullosa herpetiformis,Epilepsy, Episodic ataxia, Erythrokeratodermia variabilis,Erythropoietic protoporphyria, Exercise intolerance, Exudativevitreoretinopathy, Fabry disease, Factor V deficiency, Factor VIIdeficiency, Factor xiii deficiency, Familial adenomatous polyposis,breast cancer, ovarian cancer, cold urticarial, chronic infantileneurological, cutaneous and articular syndrome, hemiplegic migraine,hypercholesterolemia, hypertrophic cardiomyopathy,hypoalphalipoproteinemia, hypokalemia-hypomagnesemia, juvenile gout,hyperlipoproteinemia, visceral amyloidosis, hypophosphatemic vitamin Drefractory rickets, FG syndrome, Fibrosis of extraocular muscles,Finnish congenital nephrotic syndrome, focal epilepsy, Focal segmentalglomerulosclerosis, Frontonasal dysplasia, Frontotemporal dementia,Fructose-biphosphatase deficiency, Gamstorp-Wohlfart syndrome,Ganglioside sialidase deficiency, GATA-I -related thrombocytopenia,Gaucher disease, Giant axonal neuropathy, Glanzmann thrombasthenia,Glomerulocystic kidney disease, Glomerulopathy, Glucocorticoidresistance, Glucose-6-phosphate transport defect, Glutaric aciduria,Glycogen storage disease, Gorlin syndrome, Holoprosencephaly, GRACILEsyndrome, Haemorrhagic telangiectasia, Hemochromatosis, Hemoglobin Hdisease, Hemolytic anemia, Hemophagocytic lymphohistiocytosis, Carcinomaof colon, Myhre syndrome, leukoencephalopathy, Hereditary factor IXdeficiency disease, Hereditary factor VIII deficiency disease,Hereditary factor XI deficiency disease, Hereditary fructosuria,Hereditary Nonpolyposis Colorectal Neoplasm, Hereditary pancreatitis,Hereditary pyropoikilocytosis, Elliptocytosis, Heterotaxy, Heterotopia,Histiocytic medullary reticulosis, Histiocytosis-lymphadenopathy plussyndrome, HNSHA due to aldolase A deficiency, Holocarboxylase synthetasedeficiency, Homocysteinemia, Rowel-Evans syndrome, Hydatidiform mole,Hypercalciuric hypercalcemia, Hyperimmunoglobulin D, Mevalonic aciduria,Hyperinsulinemic hypoglycemia, Hyperkalemic Periodic Paralysis,Paramyotonia congenita of von Eulenburg, Hyperlipoproteinemia,Hypermanganesemia, Hypermethioninemia, Hyperphosphatasemia,Hypertension, hypomagnesemia, Hypobetalipoproteinemia, Hypocalcemia,Hypogonadotropic hypogonadism, Hypogonadotropic hypogonadism,Hypohidrotic ectodermal dysplasia, Hyper-IgM immunodeficiency,Hypohidrotic X-linked ectodermal dysplasia, Hypomagnesemia,Hypoparathyroidism, Idiopathic fibrosing alveolitis, Immunodeficiency,Immunoglobulin A deficiency, Infantile hypophosphatasia, InfantileParkinsonism-dystonia, Insulin-dependent diabetes mellitus, Intermediatemaple syrup urine disease, Ischiopatellar dysplasia, Islet cellhyperplasia, Isolated growth hormone deficiency, Isolated lutropindeficiency, Isovaleric acidemia, Joubert syndrome, Juvenile polyposissyndrome, Juvenile retinoschisis, Kallmann syndrome, Kartagenersyndrome, Kugelberg-W elander disease, Lattice corneal dystrophy, Lebercongenital amaurosis, Leber optic atrophy, Left ventricularnoncompaction, Leigh disease, Mitochondrial complex I deficiency,Leprechaunism syndrome, Arthrogryposis, Anterior horn cell disease,Leukocyte adhesion deficiency, Leukodystrophy, Leukoencephalopathy,Ovarioleukodystrophy, L-ferritin deficiency, Li-Fraumeni syndrome,Limb-girdle muscular dystrophy-dystroglycanopathy, Loeys-Dietz syndrome,Long QT syndrome, Macrocephaly/autism syndrome, Macular cornealdystrophy, Macular dystrophy, Malignant hyperthermia susceptibility,Malignant tumor of prostate, Maple syrup urine disease, Marden Walkerlike syndrome, Marfan syndrome, Marie Unna hereditary hypotrichosis,Mast cell disease, Meconium ileus, Medium-chain acyl-coenzyme Adehydrogenase deficiency, Melnick-Fraser syndrome, Mental retardation,Merosin deficient congenital muscular dystrophy, Mesothelioma,Metachromatic leukodystrophy, Metaphyseal chondrodysplasia,Methemoglobinemia, methylmalonic aciduria, homocystinuria, Microcephaly,chorioretinopathy, lymphedema, Microphthalmia, Mild non-PKUhyperphenylalanemia, Mitchell-Riley syndrome, mitochondrial3-hydroxy-3-methylglutaryl-CoA synthase deficiency, Mitochondrialcomplex I deficiency, Mitochondrial complex III deficiency,Mitochondrial myopathy, Mucolipidosis III, Mucopolysaccharidosis,Multiple sulfatase deficiency, Myasthenic syndrome, Mycobacteriumtuberculosis, Myeloperoxidase deficiency, Myhre syndrome, Myoclonicepilepsy, Myofibrillar myopathy, Myoglobinuria, Myopathy, Myopia,Myotonia congenital, Navajo neurohepatopathy, Nemaline myopathy,Neoplasm of stomach, Nephrogenic diabetes insipidus, Nephronophthisis,Nephrotic syndrome, Neurofibromatosis, Neutral lipid storage disease,Niemann-Pick disease, Non-ketotic hyperglycinemia, Noonan syndrome,Noonan syndrome-like disorder, Norum disease, Macular degeneration,N-terminal acetyltransferase deficiency, Oculocutaneous albinism,Oculodentodigital dysplasia, Ohdo syndrome, Optic nerve aplasia,Omithine carbamoyltransferase deficiency, Orofaciodigital syndrome,Osteogenesis imperfecta, Osteopetrosis, Ovarian dysgenesis,Pachyonychia, Palmoplantar keratoderma, nonepidermolytic,Papillon-Lef\xc3\xa8vre syndrome, Haim-Munk syndrome, Periodontitis,Peeling skin syndrome, Pendred syndrome, Peroxisomal fatty acyl-coareductase I disorder, Peroxisome biogenesis disorder, Pfeiffer syndrome,Phenylketonuria, Phenylketonuria, Hyperphenylalaninemia, non-PKU,Pituitary hormone deficiency, Pityriasis rubra pilaris, Polyarteritisnodosa, Polycystic kidney disease, Polycystic lipomembranousosteodysplasia, Polymicrogyria, Pontocerebellar hypoplasia,Porokeratosis, Posterior column ataxia, Primary erythromelalgia,hyperoxaluria, Progressive familial intrahepatic cholestasis,Progressive pseudorheumatoid dysplasia, Propionic acidemia,Pseudohermaphroditism, Pseudohypoaldosteronism, Pseudoxanthomaelasticum-like disorder, Purine-nucleoside phosphorylase deficiency,Pyridoxal 5-phosphate-dependent epilepsy, Renal dysplasia, retinalpigmentary dystrophy, cerebellar ataxia, skeletal dysplasia, Reticulardysgenesis, Retinitis pigmentosa, Usher syndrome, Retinoblastoma,Retinopathy, RRM2B-related mitochondrial disease, Rubinstein-Taybisyndrome, Schnyder crystalline corneal dystrophy, Sebaceous tumor,Severe congenital neutropenia, Severe myoclonic epilepsy in infancy,Severe X-linked myotubular myopathy, onychodysplasia, facialdysmorphism, hypotrichosis, Short-rib thoracic dysplasia, Sialic acidstorage disease, Sialidosis, Sideroblastic anemia, Small fiberneuropathy, Smith-Magenis syndrome, Sorsby fundus dystrophy, Spasticataxia, Spastic paraplegia, Spermatogenic failure, Spherocytosis,Sphingomyelin/cholesterol lipidosis, Spinocerebellar ataxia,Split-hand/foot malformation, Spondyloepimetaphyseal dysplasia,Platyspondylic lethal skeletal dysplasia, Squamous cell carcinoma of thehead and neck, Stargardt disease, Sucrase-isomaltase deficiency, Suddeninfant death syndrome, Supravalvar aortic stenosis, Surfactantmetabolism dysfunction, Tangier disease, Tatton-Brown-rahman syndrome,Thoracic aortic aneurysms and aortic dissections, Thrombophilia, Thyroidhormone resistance, TNF receptor-associated periodic fever syndrome(TRAPS), Tooth agenesis, Torsades de pointes, Transposition of greatarteries, Treacher Collins syndrome, Tuberous sclerosis syndrome,Tyrosinase-negative oculocutaneous albinism, Tyrosinase-positiveoculocutaneous albinism, Tyrosinemia, UDPglucose-4-epimerase deficiency,Ullrich congenital muscular dystrophy, Bethlem myopathy Usher syndrome,UV-sensitive syndrome, Van der Woude syndrome, popliteal pterygiumsyndrome, Very long chain acyl-CoA dehydrogenase deficiency,Vesicoureteral reflux, Vitreoretinochoroidopathy, Von Rippel-Lindausyndrome, von Willebrand disease, Waardenburg syndrome, Warsaw breakagesyndrome, WFSI -Related Disorders, Wilson disease, Xerodermapigmentosum, X-linked agammaglobulinemia, X-linked hereditary motor andsensory neuropathy, X-linked severe combined immunodeficiency, andZellweger syndrome.

In certain embodiments, the methods, systems, and compositions describedherein are used to correct one or more pathogenic T(U)-to-C or A-to-Gmutations/SNPs as provided in the Table below.

TABLE 7 Candidate Gene Disease NM_007262.4(PARK7): c.497T > C PARK7Parkinson disease 7 (p.Leu166Pro) NM_174936.3(PCSK9): c.646T > C PCSK9Hypercholesterolemia, (p.Phe216Leu) autosomal dominant, 3NM_000642.2(AGL): c.3083 + 2T > C AGL Glycogen storage disease type IIINM_213653.3(HFE2): c.842T > C HFE2 Hemochromatosis type 2A (p.Ile281Thr)NM_170707.3(LMNA): c.799T > C LMNA Primary dilated (p.Tyr267His)cardiomyopathy|not provided NM_000488.3(SERPINC1): c.1141T > C SERPINC1Antithrombin III deficiency (p.Ser381Pro) NM_000465.3(BARD1): c.1159T >C BARD1 Familial cancer of breast|not (p.Phe387Leu) specified|Hereditarycancer- predisposing syndrome NM_000030.2(AGXT): c.613T > C AGXT Primaryhyperoxaluria, type (p.Ser205Pro) I|not provided NM_001302946.1(TRNT1):c.668T > C TRNT1 Sideroblastic anemia with B- (p.Ile223Thr) cellimmunodeficiency, periodic fevers, and developmental delayNM_138694.3(PKHD1): c.8068T > C PKHD1 Autosomal recessive (p.Trp2690Arg)polycystic kidney disease NM_000162.3(GCK): c.1169T > C GCKMaturity-onset diabetes of the (p.IIe390Thr) young, type 2NM_017890.4(VPS13B): c.7504 + 2T > C VPS13B Cohen syndromeNM_000155.3(GALT): c.512T > C GALT Deficiency of UDPglucose-(p.Phe171Ser) hexose-1-phosphate uridylyltransferase NM_000277.1 (PAH):c.691T > C PAH Phenylketonuria|not provided (p.Ser231Pro)NM_000138.4(FBN1): c.4531T > C FBN1 Marfan syndrome (p.Cys1511Arg)NM_000527.4(LDLR): c.1745T > C LDLR Familial hypercholesterolemia(p.Leu582Pro)

Some exemplary embodiments are described in the following numberedparagraphs.

-   1. An engineered, non-naturally occurring system suitable for    modifying a nucleic acid at target loci, comprising a) a targeting    component or a nucleotide sequence encoding the targeting component    and b) a base editing component or a nucleotide sequence encoding    the base editing component.-   2. The system of paragraph 1, wherein the targeting domain is a    DNA-binding protein or functional portion thereof, or a RNA-binding    protein or functional portion thereof.-   3. The system of paragraph 2, wherein the DNA-binding protein is    Cas9 or Cpf1 and the system further comprises a guide molecule which    comprises a guide sequence, or a nucleotide encoding said guide    molecule.-   4. The system of paragraph 2, wherein the RNA-binding protein is a    Cas13 protein and the system further comprises a guide molecule    comprising guide sequence or a nucleotide sequence encoding said    guide molecule.-   5. The system of paragraphs 3 or 4, wherein the Cas9, Cpf1, or Cas13    is a dead Cas9, dead Cpf1, or dead Cas13.-   6. The system of any one of the proceeding paragraphs wherein the    base editing component is adenosine deaminse or catalytic domain    thereof.-   7. The system of paragraph 1, wherein the adenosine deaminase or    catalytic domain thereof is fused to the targeting component.-   8. The system of paragraph 6, wherein the adenosine deaminase or    catalytic domain thereof is fused to the targeting component by a    linker.-   9. The system of any one of the preceding paragraphs, wherein said    adenosine deaminase protein or catalytic domain thereof is a human,    cephalopod, or Drosophila adenosine deaminase protein or catalytic    domain thereof.-   10. The system of any one of the proceeding paragraphs, wherein said    adenosine deaminase protein or catalytic domain thereof has been    modified to comprise a mutation at glutamic acid488 of the hADAR2-D    amino acid sequence, or a corresponding position in a homologous    ADAR protein.-   11. The system of paragraphs 10, wherein said glutamic acid residue    at position 488 or a corresponding position in a homologous ADAR    protein is replaced by a glutamine residue (E488Q).-   12. The system of any one of the proceeding paragraphs wherein said    adenosine deaminase protein or catalytic domain thereof is a mutated    hADAR2d comprising mutation E488Q or a mutated hADAR1d comprising    mutation E1008Q.-   13. A vector system comprising one or more vectors encoding the    systems of any one of the proceeding paragraphs.-   14. An in vitro or ex vivo host cell or progeny thereor or cell line    or progeny thereof comprising the system of any one of paragraphs 1    to 12.-   15. A method for programmable and targeted base editing of tarcet    loci comprising delivery of the system of any one of paragraphs    1-12.-   16. The method of paragraph 15, wherein said method comprises,    determining said target sequence of interest and selecting said    adenosine deaminase protein or catalytic domain thereof which most    efficiently deaminates said Adenine present in said target sequence.-   17. The method of paragraph 16, wherein said deamination of said    Adenine in said target RNA of interest remedies a disease caused by    transcripts containing a pathogenic G→A or C→T point mutation.-   18. The method of paragraph 17, wherein said disease is selected    from Meier-Gorlin syndrome, Seckel syndrome 4, Joubert syndrome 5,    Leber congenital amaurosis 10; Charcot-Marie-Tooth disease, type 2;    Charcot-Marie-Tooth disease, type 2; Usher syndrome, type 2C;    Spinocerebellar ataxia 28; Spinocerebellar ataxia 28;    Spinocerebellar ataxia 28; Long QT syndrome 2; Sjögren-Larsson    syndrome; Hereditary fructosuria; Hereditary fructosuria;    Neuroblastoma; Neuroblastoma; Kallmann syndrome 1; Kallmann syndrome    1; Kallmann syndrome 1; Metachromatic leukodystrophy, Rett syndrome,    Amyotrophic lateral sclerosis type 10, Li-Fraumeni syndrome, or a    disease listed in Table 5.

The invention is further described in the following examples, which donot limit the scope of the invention described in the claims.

EXAMPLES Example 1

Adenine deaminases (ADs) is capable of deaminating adenines at specificsites in double stranded RNA.

The facts that some ADs can effect adenine deamination on DNA-RNAn RNAduplexes (e.g. Zheng et al., Nucleic Acids Research 2017) presents aunique opportunity to develop an RNA guided AD by taking advantage ofthe RNA duplex formed between the guide RNA and its complementary DNAtarget in the R-loop formed during RNA-guided DNA binding by inactiveCas13. By using inactive Cas13 to recruit an AD, the AD enzyme will thenact on the adenine in the RNA-DNAn RNA duplex.

In one embodiment, an inactive Cas13, such as Cas13b is obtained usingthe following mutations: R116A, H121A, R1177A and H1182A. To increasethe efficiency of editing by AD, a mutated ADAR is used such as themutated hADAR2d comprising mutation E488Q.

Designs for the Recruitment of AD to a Specific Locus:

1. NLS-tagged inactive Cas13 is fused to AD on either the N- orC-terminal end. A variety of linkers are used including flexible linkerssuch as GSGS (SEQ ID NO: 10) or less flexible linkers such asLEPGEKPYKCPECGKSFSQSGALTRHQRTHTR.

2. The guide RNA scaffold is modified with aptamers such as MS2 bindingsites (e.g. Konermann et al., Nature 2015). NLS-tagged AD-MS2 bindingprotein fusions is co-introduced into target cells along with(NLS-tagged inactive or Cas13b) and corresponding guide RNA.

3. AD is inserted into an internal loop of NLS-tagged inactive ornickase Cas13.

Designs for the RNA Guide:

1. Guide sequences of a length corresponding to that of a natural guidesequence of the Cas13 protein are designed to target the RNA ofinterest.

2. RNA guide with longer than canonical length is used to form RNAduplexes outside of the protein-guide RNA-target DNA complex.

For each of these RNA guide designs, the base on the RNA that isopposite of the adenine on the target RNA strand would be specified as aC as opposed to U.

Choice and Designs of ADs:

A number of ADs are used, and each will have varying levels of activity.These ADs

1. Human ADARs (hADAR1, hADAR2, hADAR3)

2. Squid Octopus vulgaris ADARs

3. Squid Sepia ADARS; Doryteusthis opalescens ADARS

ADATs (human ADAT, Drosophila ADAT)

Mutations can also be used to increase the activity of ADAR reactingagainst a DNA-RNAn RNA duplex. For example, for the human ADAR genes,the hADAR1d(E1008Q) or hADAR2d(E488Q) mutation is used to increase theiractivity against a DNA-RNA target.

Each ADAR has varying levels of sequence context requirement. Forexample, for hADAR1d (E1008Q), tAg and aAg sites are efficientlydeaminated, whereas aAt and cAc are less efficiently edited, and gAa andgAc are even less edited. However, the context requirement will vary fordifferent ADARs.

A schematic showing of one version of the system is provided in FIG. 1.The amino acid sequences of example AD proteins are provided in FIG. 4.

The embodiments, illustratively described herein may suitably bepracticed in the absence of any element or elements, limitation orlimitations, not specifically disclosed herein. Thus, for example, theterms “comprising,” “including,” “containing,” etc. shall be readexpansively and without limitation. Additionally, the terms andexpressions employed herein have been used as terms of description andnot of limitation, and there is no intention in the use of such termsand expressions of excluding any equivalents of the features shown anddescribed or portions thereof, but it is recognized that variousmodifications are possible within the scope of the claimed technology.Additionally, the phrase “consisting essentially of” will be understoodto include those elements specifically recited and those additionalelements that do not materially affect the basic and novelcharacteristics of the claimed technology. The phrase “consisting of”excludes any element not specified.

The present disclosure is not to be limited in terms of the particularembodiments described in this application. Many modifications andvariations can be made without departing from its spirit and scope, aswill be apparent to those skilled in the art. Functionally equivalentmethods and compositions within the scope of the disclosure, in additionto those enumerated herein, will be apparent to those skilled in the artfrom the foregoing descriptions. Such modifications and variations areintended to fall within the scope of the appended claims. The presentdisclosure is to be limited only by the terms of the appended claims,along with the full scope of equivalents to which such claims areentitled. It is to be understood that this disclosure is not limited toparticular methods, reagents, compounds compositions or biologicalsystems, which can of course vary. It is also to be understood that theterminology used herein is for the purpose of describing particularembodiments only, and is not intended to be limiting.

In addition, where features or aspects of the disclosure are describedin terms of Markush groups, those skilled in the art will recognize thatthe disclosure is also thereby described in terms of any individualmember or subgroup of members of the Markush group.

As will be understood by one skilled in the art, for any and allpurposes, particularly in terms of providing a written description, allranges disclosed herein also encompass any and all possible subrangesand combinations of subranges thereof. Any listed range can be easilyrecognized as sufficiently describing and enabling the same range beingbroken down into at least equal halves, thirds, quarters, fifths,tenths, etc. As a non-limiting example, each range discussed herein canbe readily broken down into a lower third, middle third and upper third,etc. As will also be understood by one skilled in the art all languagesuch as “up to,” “at least,” “greater than,” “less than,” and the like,include the number recited and refer to ranges which can be subsequentlybroken down into subranges as discussed above. Finally, as will beunderstood by one skilled in the art, a range includes each individualmember.

All publications, patent applications, issued patents, and otherdocuments referred to in this specification are herein incorporated byreference as if each individual publication, patent application, issuedpatent, or other document was specifically and individually indicated tobe incorporated by reference in its entirety. Definitions that arecontained in text incorporated by reference are excluded to the extentthat they contradict definitions in this disclosure.

Example 2 Cluc/Gluc Tiling for Cas13a/Cas13b Interference

To compare knockdown efficiency between Cas13a and Cas13b, Cypridina andGaussia luciferase genes were tiled with 24 or 96 guides, respectively(FIG. 10). Guides were matched for Cas13a and Cas13b, and show increasedknockdown efficiency for Cas13b, with all but one guide for each geneshowing higher efficiency for Cas13b.

ADAR Editing Quantification by NGS

Cas13b-ADAR2 RNA editing efficiency was tested by designing a luciferasereporter with a premature stop codon UAG, which prevents expression ofthe luciferase (FIG. 11A). 7 guides of varying length were designed andpositioned relative to the UAG stop codon that all contained a Cmismatch to the A in the UAG. The C mismatch is known to create a bubbleat the site of editing which is favored by the ADAR catalytic domain.RNA editing by Cas13b-ADAR2 would convert the UAG to a UIG (UGG), whichintroduces a tryptophan instead of the stop codon, and allowstranslation to proceed. Expression of the guides and Cas13b12-ADAR2fusion in HEK293FT cells restored luciferase expression to varyinglevels with the greatest restoration occurring for guide 5 (FIG. 11B).In general, there is increasing levels of editing from guides 1-5 as theediting site is moved further away from the 3′ end of the crRNA wherethe direct repeat is and thus where the protein binds. This likelyindicates that the part of the crRNA:target duplex that is bound by theprotein is inaccessible to the ADAR catalytic domain. Guides 5, 6, and 7show the greatest amount of activity because the editing site is on thefar end of the guide away from the DR/protein binding area and becausetheir guides are much longer, generating a longer RNA duplex that isfavored by ADAR. ADAR activity is optimal when the editing site is inthe middle of a RNA duplex. The relative expression of luciferaseactivity is normalized to the non-targeting guide condition.

These samples were sequenced to precisely quantitate the RNA editingefficiency (FIG. 11C). The editing efficiency is listed in parenthesesnext to the guide label. Overall, the percent editing identified bysequencing matched the relative levels of luciferase expressionrestoration seen in FIG. 11B. Guide 5 showed the most RNA editing with arate of 45% conversion to G at the on-target A. In some instances, thereis a small amount of off-target A-G editing in the region. These may bereduced by introducing G mismatches in the guide sequence, which aredisfavored by the ADAR catalytic domain.

In addition to editing the luciferase reporter transcript, guides weredesigned to edit out-of-frame UAG sites in the KRAS and PPIBtranscripts, with two guides targeting each transcript (FIG. 12). Theguides were designed with the same principles as guide 5 above (a 45 ntspacer with the editing site 27 nt away from the 3′ DR and a C mismatchto the editing site adenosine). The KRAS guides were able to achieve6.5% and 13.7% editing at the on-target adenosine and the PPIB guideswere able to achieve 7.7% and 9.2% editing. There are also someoff-targets present for some of these guides which can be reduced bydesigning G mismatches in the spacers against possible off-targetadenosines that are nearby. It does seem that off-targets seem to happenwith the duplex region 3′ of the target adenosine.

Cas13a/b+shRNA Specificity from RNA Seq

To determine the specificity of the Cas13b12 knockdown, RNA sequencingwas performed on all mRNAs across the transcriptome (FIG. 13A). Theknockdown of guides targeting Gluc and KRAS was compared againstnon-targeting guides and found that Cas13a2 and Cas13b12 had specificknockdown of the target transcript (red dot in FIG. 13A) while theshRNAs had many off-targets as evidenced by the greater variance in thedistribution. The number of significant off-targets for each of theseconditions is shown in FIG. 13B. Significant off-targets are measured bya t-test with FDR correction (p<0.01) for any off-target transcriptsthat are changed by greater than 2 fold or less than 0.8 fold. TheCas13a and Cas13b conditions had very few off targets compared to thehundreds of off-targets found for the shRNA conditions. The knockdownefficiency for each of the conditions is shown in FIG. 13C.

Mismatch Specificity to Reduce Off Targets (A:A or A:G)

To reduce off targets at adenosines near the target adenosine editingsite, guides were designed that have G or A mismatches to the potentialoff-target adenosines (FIG. 14 and Table below). Mismatches with G or Aare not favored for activity by the ADAR catalytic domain.

TABLE 8 Name Guide Luciferase guide WT with C mismatchcatagaatgttctaaaCCAtcctgcggcctctactctgcattcaa (SEQ ID NO: 162)Luciferase guide WT with C mismatch with 1catagaatgttcGaaaCCAtcctgcggcctctactctgcattcaa G MM (SEQ ID NO: 163)Luciferase guide WT with C mismatch with 2catagaatgGtcGaaaCCAtcctgcggcctctactctgcattcaa G MM (SEQ ID NO: 164)Luciferase guide WT with C mismatch with 1catagaatgttcAaaaCCAtcctgcggcctctactctgcattcaa G MM (SEQ ID NO: 165)Luciferase guide WT with C mismatch with 2catagaatgAtcAaaaCCAtcctgcggcctctactctgcattcaa G MM (SEQ ID NO: 166)KRAS guide WT with Cmismatch (SEQ IDggtttctccatcaattacCacttgcttcctgtaggaatcctctatt NO: 167)KRAS guide with C mismatch with 1 G MMggtttctccatcaatGacCacttgcttcctgtaggaatcctctatt (SEQ ID NO: 168)KRAS guide with C mismatch with 2 G MMggtttctccatcaaGGacCacttgcttcctgtaggaatcctctatt (SEQ ID NO: 169)KRAS guide with C mismatch with 1 A MMggtttctccatcaatAacCacttgcttcctgtaggaatcctctatt (SEQ ID NO: 170)KRAS guide with C mismatch with 2 A MMggtttctccatcaaAAacCacttgcttcctgtaggaatcctctatt (SEQ ID NO: 171)PPIB guide WT with C mismatch (SEQ IDgcctttctctcctgtagcCaaggccacaaaattatccactgttttt NO: 172)PPIB guide WT with C mismatch with 1 GgcctttctctcctgGagcCaaggccacaaaattatccactgttttt MM (SEQ ID NO: 173)PPIB guide WT with C mismatch with 1 AgcctttctctcctgAagcCaaggccacaaaattatccactgttttt MM (SEQ ID NO: 174)

The guides in the Table above were designed to have a C mismatch againstthe on-target adenosine to be edited and G or A mismatches against knownoff-target sites (based off of the RNA sequencing from above).Mismatches in the spacer sequence are capitalized.

Mismatch for On-Target Activity

Prior research on the catalytic domain of ADAR2 has demonstrated thatdifferent bases opposite the target A can influence the amount ofinosine editing (Zheng et al. (2017), Nucleic Acid Research,45(6):3369-3377). Specifically, U and C are found opposite naturalADAR-edited A's, whereas G and A are not. To test whether or not A and Gmismatches with the edited A can be used to suppress ADAR activity aguide known to be active with a C mismatch is tested with all other 3possible bases on the luciferase reporter assay (FIG. 16). Relativeactivities are quantified by assessing luciferase activity. Guidesequences are provided in the Table below.

TABLE 9 Mismatch Guide sequence Mismatch-CGcatagaatgttctaaaCCAtcctgcggcctct (SEQ ID NO: 175) actctgcattcaaMismatch-G GcatagaatgttctaaaCGAtcctgcggcctct (SEQ ID NO: 176)actctgcattcaa Mismatch-T GcatagaatgttctaaaCTAtcctgcggcctct(SEQ ID NO: 177) actctgcattcaa Mismatch-AGcatagaatgttctaaaCAAtcctgcggcctct (SEQ ID NO: 178) actctgcattcaaImprovement of Editing and Reduction of Off-Target Modification byChemical Modification of gRNAs

gRNAs which are chemically modified as exemplified in Vogel et al.(2014), Angew Chem Int Ed, 53:6267-6271, doi:10.1002/anie.201402634) toreduce off-target activity and to improve on-target efficiency.2′-O-methyl and phosphothioate modified guide RNAs in general improveediting efficiency in cells.

Motif Preference

ADAR has been known to demonstrate a preference for neighboringnucleotides on either side of the edited A(www.nature.com/nsmb/journal/v23/n5/full/nsmb.3203.html, Matthews et al.(2017), Nature Structural Mol Biol, 23(5): 426-433). The preference issystematically tested by targeting Cypridina luciferase transcripts withvariable bases surrounding the targeted A (FIG. 17).

Larger Bubbles to Enhance RNA Editing Efficiency

To enhance RNA editing efficiency on non-preferred 5′ or 3′ neighboringbases, intentional mismatches in neighboring bases are introduced, whichhas been demonstrated in vitro to allow for editing of non-preferredmotifs (academic.oup.com/nar/article-lookup/doi/10.1093/nar/gku272;Schneider et al (2014), Nucleic Acid Res, 42(10):e87); Fukuda et al.(2017), Scienticic Reports, 7, doi:10.1038/srep41478). Additionalmismatches are tested, such as guanosine substitutions, to see if theyreduce natural preferences (FIG. 18).

Editing of Multiple A's in a Transcript

Results suggest that As opposite Cs in the targeting window of the ADARdeaminase domain are preferentially edited over other bases.Additionally, As base-paired with Us within a few bases of the targetedbase show low levels of editing by Cas13b-ADAR fusions, suggesting thatthere is flexibility for the enzyme to edit multiple As (FIG. 19). Thesetwo observations suggest that multiple As in the activity window ofCas13b-ADAR fusions could be specified for editing by mismatching all Asto be edited with Cs. To test this the most promising guides from theoptimization experiment are taken and multiple A:C mismatches in theactivity window are designed to test the possibility of creatingmultiple A:I edits. The editing rates for this experiment is bequanitifed using NGS. To suppress potential off-target editing in theactivity window, non-target As are paired with As or Gs (depending onthe results from the base preference experiment).

Guide Length Titration for RNA Editing

ADAR naturally works on inter- or intra-molecular RNA duplexes of >20 bpin length (see also Nishikura et al. (2010), Annu Rev Biochem,79:321-349). The results demonstrated that longer crRNAs, resulting inlonger duplexes, had higher levels of activity. To systematicallycompare the activity of guides of different lengths for RNA editingactivity we have designed guides of 30, 50, 70 and 84 bases to correctthe stop codon in our luciferase reporter assay (FIGS. 20A-20F and Tablebelow). We have designed these guides such that the position of theedited A is present at all possible even distances within the mRNA:crRNAduplex with respect to the 3′ end of the specificity determining regionof the crRNA (i.e. +2, +4 etc.).

TABLE 10 Sequence Name Sequence Guide_Cas13bC-GCATCCTGCGGCCTCTACTCTGCATTCAATT luc_30_ADAR0Top (SEQ ID NO: 179)Guide_Cas13bC- GACCATCCTGCGGCCTCTACTCTGCATTCAA luc_30_ADAR1Top(SEQ ID NO: 180) Guide_Cas13bC- GAAACCATCCTGCGGCCTCTACTCTGCATTCluc_30_ADAR2Top (SEQ ID NO: 181) Guide_Cas13bC-GCTAAACCATCCTGCGGCCTCTACTCTGCAT luc_30_ADAR3Top (SEQ ID NO: 182)Guide_Cas13bC- GTTCTAAACCATCCTGCGGCCTCTACTCTGC luc_30_ADAR4Top(SEQ ID NO: 183) Guide_Cas13bC- GTGTTCTAAACCATCCTGCGGCCTCTACTCTluc_30_ADAR5Top (SEQ ID NO: 184) Guide_Cas13bC-GAATGTTCTAAACCATCCTGCGGCCTCTACT luc_30_ADAR6Top (SEQ ID NO: 185)Guide_Cas13bC- GAGAATGTTCTAAACCATCCTGCGGCCTCTA luc_30_ADAR7Top(SEQ ID NO: 186) Guide_Cas13bC- GATAGAATGTTCTAAACCATCCTGCGGCCTCluc_30_ADAR8Top (SEQ ID NO: 187) Guide_Cas13bC-GCCATAGAATGTTCTAAACCATCCTGCGGCC luc_30_ADAR9Top (SEQ ID NO: 188)Guide_Cas13bC- GTTCCATAGAATGTTCTAAACCATCCTGCGG luc_30_ADAR10Top(SEQ ID NO: 189) Guide_Cas13bC- GCTTTCCATAGAATGTTCTAAACCATCCTGCluc_30_ADAR11Top (SEQ ID NO: 190) Guide_Cas13bC-GCTCTTTCCATAGAATGTTCTAAACCATCCT luc_30_ADAR12Top (SEQ ID NO: 191)Guide_Cas13bC- GATCTCTTTCCATAGAATGTTCTAAACCATC luc_30_ADAR13Top(SEQ ID NO: 192) Guide_Cas13bC- GGAATCTCTTTCCATAGAATGTTCTAAACCAluc_30_ADAR14Top (SEQ ID NO: 193) Guide_Cas13bC-GCATCCTGCGGCCTCTACTCTGCATTCAATTACATACTGACA luc_50_ADAR0Top CATTCGGCA(SEQ ID NO: 194) Guide_Cas13bC-GACCATCCTGCGGCCTCTACTCTGCATTCAATTACATACTGA luc_50_ADAR1Top CACATTCGG(SEQ ID NO: 195) Guide_Cas13bC-GAAACCATCCTGCGGCCTCTACTCTGCATTCAATTACATACT luc_50_ADAR2Top GACACATTC(SEQ ID NO: 196) Guide_Cas13bC-GCTAAACCATCCTGCGGCCTCTACTCTGCATTCAATTACATA luc_50_ADAR3Top CTGACACAT(SEQ ID NO: 197) Guide_Cas13bC-GTTCTAAACCATCCTGCGGCCTCTACTCTGCATTCAATTACAT luc_50_ADAR4Top ACTGACAC(SEQ ID NO: 198) Guide_Cas13bC-GTGTTCTAAACCATCCTGCGGCCTCTACTCTGCATTCAATTAC luc_50_ADAR5Top ATACTGAC(SEQ ID NO: 199) Guide_Cas13bC-GAATGTTCTAAACCATCCTGCGGCCTCTACTCTGCATTCAATT luc_50_ADAR6Top ACATACTG(SEQ ID NO: 200) Guide_Cas13bC-GAGAATGTTCTAAACCATCCTGCGGCCTCTACTCTGCATTCA luc_50_ADAR7Top ATTACATAC(SEQ ID NO: 201) Guide_Cas13bC-GATAGAATGTTCTAAACCATCCTGCGGCCTCTACTCTGCATT luc_50_ADAR8Top CAATTACAT(SEQ ID NO: 202) Guide_Cas13bC-GCCATAGAATGTTCTAAACCATCCTGCGGCCTCTACTCTGCA luc_50_ADAR9Top TTCAATTAC(SEQ ID NO: 203) Guide_Cas13bC-GTTCCATAGAATGTTCTAAACCATCCTGCGGCCTCTACTCTG luc_50_ADAR10Top CATTCAATT(SEQ ID NO: 204) Guide_Cas13bC-GCTTTCCATAGAATGTTCTAAACCATCCTGCGGCCTCTACTCT luc_50_ADAR11Top GCATTCAA(SEQ ID NO: 205) Guide_Cas13bC-GCTCTTTCCATAGAATGTTCTAAACCATCCTGCGGCCTCTACT luc_50_ADAR12Top CTGCATTC(SEQ ID NO: 206) Guide_Cas13bC-GATCTCTTTCCATAGAATGTTCTAAACCATCCTGCGGCCTCTA luc_50_ADAR13Top CTCTGCAT(SEQ ID NO: 207) Guide_Cas13bC-GGAATCTCTTTCCATAGAATGTTCTAAACCATCCTGCGGCCT luc_50_ADAR14Top CTACTCTGC(SEQ ID NO: 208) Guide_Cas13bC-GTGGAATCTCTTTCCATAGAATGTTCTAAACCATCCTGCGGC luc_50_ADAR15Top CTCTACTCT(SEQ ID NO: 209) Guide_Cas13bC-GACTGGAATCTCTTTCCATAGAATGTTCTAAACCATCCTGCG luc_50_ADAR16Top GCCTCTACT(SEQ ID NO: 210) Guide_Cas13bC-GGAACTGGAATCTCTTTCCATAGAATGTTCTAAACCATCCTG luc_50_ADAR17Top CGGCCTCTA(SEQ ID NO: 211) Guide_Cas13bC-GTGGAACTGGAATCTCTTTCCATAGAATGTTCTAAACCATCC luc_50_ADAR18Top TGCGGCCTC(SEQ ID NO: 212) Guide_Cas13bC-GCCTGGAACTGGAATCTCTTTCCATAGAATGTTCTAAACCAT luc_50_ADAR19Top CCTGCGGCC(SEQ ID NO: 213) Guide_Cas13bC-GTTCCTGGAACTGGAATCTCTTTCCATAGAATGTTCTAAACC luc_50_ADAR20Top ATCCTGCGG(SEQ ID NO: 214) Guide_Cas13bC-GGGTTCCTGGAACTGGAATCTCTTTCCATAGAATGTTCTAAA luc_50_ADAR21Top CCATCCTGC(SEQ ID NO: 215) Guide_Cas13bC-GCAGGTTCCTGGAACTGGAATCTCTTTCCATAGAATGTTCTA luc_50_ADAR22Top AACCATCCT(SEQ ID NO: 216) Guide_Cas13bC-GACCAGGTTCCTGGAACTGGAATCTCTTTCCATAGAATGTTC luc_50_ADAR23Top TAAACCATC(SEQ ID NO: 217) Guide_Cas13bC-GGTACCAGGTTCCTGGAACTGGAATCTCTTTCCATAGAATGT luc_50_ADAR24Top TCTAAACCA(SEQ ID NO: 218) Guide_Cas13bC-GCATCCTGCGGCCTCTACTCTGCATTCAATTACATACTGACA luc_70_ADAR0TopCATTCGGCAACATGTTTTTCCTGGTTTAT (SEQ ID NO: 219) Guide_Cas13bC-GACCATCCTGCGGCCTCTACTCTGCATTCAATTACATACTGA luc_70_ADAR1TopCACATTCGGCAACATGTTTTTCCTGGTTT (SEQ ID NO: 220) Guide_Cas13bC-GAAACCATCCTGCGGCCTCTACTCTGCATTCAATTACATACT luc_70_ADAR2TopGACACATTCGGCAACATGTTTTTCCTGGT (SEQ ID NO: 221) Guide_Cas13bC-GCTAAACCATCCTGCGGCCTCTACTCTGCATTCAATTACATA luc_70_ADAR3TopCTGACACATTCGGCAACATGTTTTTCCTG (SEQ ID NO: 222) Guide_Cas13bC-GTTCTAAACCATCCTGCGGCCTCTACTCTGCATTCAATTACAT luc_70_ADAR4TopACTGACACATTCGGCAACATGTTTTTCC (SEQ ID NO: 223) Guide_Cas13bC-GTGTTCTAAACCATCCTGCGGCCTCTACTCTGCATTCAATTAC luc_70_ADAR5TopATACTGACACATTCGGCAACATGTTTTT (SEQ ID NO: 224) Guide_Cas13bC-GAATGTTCTAAACCATCCTGCGGCCTCTACTCTGCATTCAATT luc_70_ADAR6TopACATACTGACACATTCGGCAACATGTTT (SEQ ID NO: 225) Guide_Cas13bC-GAGAATGTTCTAAACCATCCTGCGGCCTCTACTCTGCATTCA luc_70_ADAR7TopATTACATACTGACACATTCGGCAACATGT (SEQ ID NO: 226) Guide_Cas13bC-GATAGAATGTTCTAAACCATCCTGCGGCCTCTACTCTGCATT luc_70_ADAR8TopCAATTACATACTGACACATTCGGCAACAT (SEQ ID NO: 227) Guide_Cas13bC-GCCATAGAATGTTCTAAACCATCCTGCGGCCTCTACTCTGCA luc_70_ADAR9TopTTCAATTACATACTGACACATTCGGCAAC (SEQ ID NO: 228) Guide_Cas13bC-GTTCCATAGAATGTTCTAAACCATCCTGCGGCCTCTACTCTG luc_70_ADAR10TopCATTCAATTACATACTGACACATTCGGCA (SEQ ID NO: 229) Guide_Cas13bC-GCTTTCCATAGAATGTTCTAAACCATCCTGCGGCCTCTACTCT luc_70_ADAR11TopGCATTCAATTACATACTGACACATTCGG (SEQ ID NO: 230) Guide_Cas13bC-GCTCTTTCCATAGAATGTTCTAAACCATCCTGCGGCCTCTACT luc_70_ADAR12TopCTGCATTCAATTACATACTGACACATTC (SEQ ID NO: 231) Guide_Cas13bC-GATCTCTTTCCATAGAATGTTCTAAACCATCCTGCGGCCTCTA luc_70_ADAR13TopCTCTGCATTCAATTACATACTGACACAT (SEQ ID NO: 232) Guide_Cas13bC-GGAATCTCTTTCCATAGAATGTTCTAAACCATCCTGCGGCCT luc_70_ADAR14TopCTACTCTGCATTCAATTACATACTGACAC (SEQ ID NO: 233) Guide_Cas13bC-GTGGAATCTCTTTCCATAGAATGTTCTAAACCATCCTGCGGC luc_70_ADAR15TopCTCTACTCTGCATTCAATTACATACTGAC (SEQ ID NO: 234) Guide_Cas13bC-GACTGGAATCTCTTTCCATAGAATGTTCTAAACCATCCTGCG luc_70_ADAR16TopGCCTCTACTCTGCATTCAATTACATACTG (SEQ ID NO: 235) Guide_Cas13bC-GGAACTGGAATCTCTTTCCATAGAATGTTCTAAACCATCCTG luc_70_ADAR17TopCGGCCTCTACTCTGCATTCAATTACATAC (SEQ ID NO: 236) Guide_Cas13bC-GTGGAACTGGAATCTCTTTCCATAGAATGTTCTAAACCATCC luc_70_ADAR18TopTGCGGCCTCTACTCTGCATTCAATTACAT (SEQ ID NO: 237) Guide_Cas13bC-GCCTGGAACTGGAATCTCTTTCCATAGAATGTTCTAAACCAT luc_70_ADAR19TopCCTGCGGCCTCTACTCTGCATTCAATTAC (SEQ ID NO: 238) Guide_Cas13bC-GTTCCTGGAACTGGAATCTCTTTCCATAGAATGTTCTAAACC luc_70_ADAR20TopATCCTGCGGCCTCTACTCTGCATTCAATT (SEQ ID NO: 239) Guide_Cas13bC-GGGTTCCTGGAACTGGAATCTCTTTCCATAGAATGTTCTAAA luc_70_ADAR21TopCCATCCTGCGGCCTCTACTCTGCATTCAA (SEQ ID NO: 240) Guide_Cas13bC-GCAGGTTCCTGGAACTGGAATCTCTTTCCATAGAATGTTCTA luc_70_ADAR22TopAACCATCCTGCGGCCTCTACTCTGCATTC (SEQ ID NO: 241) Guide_Cas13bC-GACCAGGTTCCTGGAACTGGAATCTCTTTCCATAGAATGTTC luc_70_ADAR23TopTAAACCATCCTGCGGCCTCTACTCTGCAT (SEQ ID NO: 242) Guide_Cas13bC-GGTACCAGGTTCCTGGAACTGGAATCTCTTTCCATAGAATGT luc_70_ADAR24TopTCTAAACCATCCTGCGGCCTCTACTCTGC (SEQ ID NO: 243) Guide_Cas13bC-GATGTACCAGGTTCCTGGAACTGGAATCTCTTTCCATAGAAT luc_70_ADAR25TopGTTCTAAACCATCCTGCGGCCTCTACTCT (SEQ ID NO: 244) Guide_Cas13bC-GGTATGTACCAGGTTCCTGGAACTGGAATCTCTTTCCATAGA luc_70_ADAR26TopATGTTCTAAACCATCCTGCGGCCTCTACT (SEQ ID NO: 245) Guide_Cas13bC-GACGTATGTACCAGGTTCCTGGAACTGGAATCTCTTTCCATA luc_70_ADAR27TopGAATGTTCTAAACCATCCTGCGGCCTCTA (SEQ ID NO: 246) Guide_Cas13bC-GACACGTATGTACCAGGTTCCTGGAACTGGAATCTCTTTCCA luc_70_ADAR28TopTAGAATGTTCTAAACCATCCTGCGGCCTC (SEQ ID NO: 247) Guide_Cas13bC-GCAACACGTATGTACCAGGTTCCTGGAACTGGAATCTCTTTC luc_70_ADAR29TopCATAGAATGTTCTAAACCATCCTGCGGCC (SEQ ID NO: 248) Guide_Cas13bC-GCCCAACACGTATGTACCAGGTTCCTGGAACTGGAATCTCTT luc_70_ADAR30TopTCCATAGAATGTTCTAAACCATCCTGCGG (SEQ ID NO: 249) Guide_Cas13bC-GGACCCAACACGTATGTACCAGGTTCCTGGAACTGGAATCTC luc_70_ADAR31TopTTTCCATAGAATGTTCTAAACCATCCTGC (SEQ ID NO: 250) Guide_Cas13bC-GTTGACCCAACACGTATGTACCAGGTTCCTGGAACTGGAATC luc_70_ADAR32TopTCTTTCCATAGAATGTTCTAAACCATCCT (SEQ ID NO: 251) Guide_Cas13bC-GCCTTGACCCAACACGTATGTACCAGGTTCCTGGAACTGGAA luc_70_ADAR33TopTCTCTTTCCATAGAATGTTCTAAACCATC (SEQ ID NO: 252) Guide_Cas13bC-GTTCCTTGACCCAACACGTATGTACCAGGTTCCTGGAACTGG luc_70_ADAR34TopAATCTCTTTCCATAGAATGTTCTAAACCA (SEQ ID NO: 253) Guide_Cas13bC-GCATCCTGCGGCCTCTACTCTGCATTCAATTACATACTGACA luc_84_ADAR0TopCATTCGGCAACATGTTTTTCCTGGTTTATTTTCACACAGTCCA (SEQ ID NO: 254)Guide_Cas13bC- GACCATCCTGCGGCCTCTACTCTGCATTCAATTACATACTGAluc_84_ADAR1Top CACATTCGGCAACATGTTTTTCCTGGTTTATTTTCACACAGTC(SEQ ID NO: 255) Guide_Cas13bC-GAAACCATCCTGCGGCCTCTACTCTGCATTCAATTACATACT luc_84_ADAR2TopGACACATTCGGCAACATGTTTTTCCTGGTTTATTTTCACACAG (SEQ ID NO: 256)Guide_Cas13bC- GCTAAACCATCCTGCGGCCTCTACTCTGCATTCAATTACATAluc_84_ADAR3Top CTGACACATTCGGCAACATGTTTTTCCTGGTTTATTTTCACAC(SEQ ID NO: 257) Guide_Cas13bC-GTTCTAAACCATCCTGCGGCCTCTACTCTGCATTCAATTACAT luc_84_ADAR4TopACTGACACATTCGGCAACATGTTTTTCCTGGTTTATTTTCAC (SEQ ID NO: 258)Guide_Cas13bC- GTGTTCTAAACCATCCTGCGGCCTCTACTCTGCATTCAATTACluc_84_ADAR5Top ATACTGACACATTCGGCAACATGTTTTTCCTGGTTTATTTTC(SEQ ID NO: 259) Guide_Cas13bC-GAATGTTCTAAACCATCCTGCGGCCTCTACTCTGCATTCAATT luc_84_ADAR6TopACATACTGACACATTCGGCAACATGTTTTTCCTGGTTTATTT (SEQ ID NO: 260)Guide_Cas13bC- GAGAATGTTCTAAACCATCCTGCGGCCTCTACTCTGCATTCAluc_84_ADAR7Top ATTACATACTGACACATTCGGCAACATGTTTTTCCTGGTTTAT(SEQ ID NO: 261) Guide_Cas13bC-GATAGAATGTTCTAAACCATCCTGCGGCCTCTACTCTGCATT luc_84_ADAR8TopCAATTACATACTGACACATTCGGCAACATGTTTTTCCTGGTTT (SEQ ID NO: 262)Guide_Cas13bC- GCCATAGAATGTTCTAAACCATCCTGCGGCCTCTACTCTGCAluc_84_ADAR9Top TTCAATTACATACTGACACATTCGGCAACATGTTTTTCCTGGT(SEQ ID NO: 263) Guide_Cas13bC-GTTCCATAGAATGTTCTAAACCATCCTGCGGCCTCTACTCTG luc_84_ADAR10TopCATTCAATTACATACTGACACATTCGGCAACATGTTTTTCCTG (SEQ ID NO: 264)Guide_Cas13bC- GCTTTCCATAGAATGTTCTAAACCATCCTGCGGCCTCTACTCTluc_84_ADAR11Top GCATTCAATTACATACTGACACATTCGGCAACATGTTTTTCC(SEQ ID NO: 265) Guide_Cas13bC-GCTCTTTCCATAGAATGTTCTAAACCATCCTGCGGCCTCTACT luc_84_ADAR12TopCTGCATTCAATTACATACTGACACATTCGGCAACATGTTTTT (SEQ ID NO: 266)Guide_Cas13bC- GATCTCTTTCCATAGAATGTTCTAAACCATCCTGCGGCCTCTAluc_84_ADAR13Top CTCTGCATTCAATTACATACTGACACATTCGGCAACATGTTT(SEQ ID NO: 267) Guide_Cas13bC-GGAATCTCTTTCCATAGAATGTTCTAAACCATCCTGCGGCCT luc_84_ADAR14TopCTACTCTGCATTCAATTACATACTGACACATTCGGCAACATGT (SEQ ID NO: 268)Guide_Cas13bC- GTGGAATCTCTTTCCATAGAATGTTCTAAACCATCCTGCGGCluc_84_ADAR15Top CTCTACTCTGCATTCAATTACATACTGACACATTCGGCAACAT(SEQ ID NO: 269) Guide_Cas13bC-GACTGGAATCTCTTTCCATAGAATGTTCTAAACCATCCTGCG luc_84_ADAR16TopGCCTCTACTCTGCATTCAATTACATACTGACACATTCGGCAAC (SEQ ID NO: 270)Guide_Cas13bC- GGAACTGGAATCTCTTTCCATAGAATGTTCTAAACCATCCTGluc_84_ADAR17Top CGGCCTCTACTCTGCATTCAATTACATACTGACACATTCGGCA(SEQ ID NO: 271) Guide_Cas13bC-GTGGAACTGGAATCTCTTTCCATAGAATGTTCTAAACCATCC luc_84_ADAR18TopTGCGGCCTCTACTCTGCATTCAATTACATACTGACACATTCGG (SEQ ID NO: 272)Guide_Cas13bC- GCCTGGAACTGGAATCTCTTTCCATAGAATGTTCTAAACCATluc_84_ADAR19Top CCTGCGGCCTCTACTCTGCATTCAATTACATACTGACACATTC(SEQ ID NO: 273) Guide_Cas13bC-GTTCCTGGAACTGGAATCTCTTTCCATAGAATGTTCTAAACC luc_84_ADAR20TopATCCTGCGGCCTCTACTCTGCATTCAATTACATACTGACACAT (SEQ ID NO: 274)Guide_Cas13bC- GGGTTCCTGGAACTGGAATCTCTTTCCATAGAATGTTCTAAAluc_84_ADAR21Top CCATCCTGCGGCCTCTACTCTGCATTCAATTACATACTGACAC(SEQ ID NO: 275) Guide_Cas13bC-GCAGGTTCCTGGAACTGGAATCTCTTTCCATAGAATGTTCTA luc_84_ADAR22TopAACCATCCTGCGGCCTCTACTCTGCATTCAATTACATACTGAC (SEQ ID NO: 276)Guide_Cas13bC- GACCAGGTTCCTGGAACTGGAATCTCTTTCCATAGAATGTTCluc_84_ADAR23Top TAAACCATCCTGCGGCCTCTACTCTGCATTCAATTACATACTG(SEQ ID NO: 277) Guide_Cas13bC-GGTACCAGGTTCCTGGAACTGGAATCTCTTTCCATAGAATGT luc_84_ADAR24TopTCTAAACCATCCTGCGGCCTCTACTCTGCATTCAATTACATAC (SEQ ID NO: 278)Guide_Cas13bC- GATGTACCAGGTTCCTGGAACTGGAATCTCTTTCCATAGAATluc_84_ADAR25Top GTTCTAAACCATCCTGCGGCCTCTACTCTGCATTCAATTACAT(SEQ ID NO: 279) Guide_Cas13bC-GGTATGTACCAGGTTCCTGGAACTGGAATCTCTTTCCATAGA luc_84_ADAR26TopATGTTCTAAACCATCCTGCGGCCTCTACTCTGCATTCAATTAC (SEQ ID NO: 280)Guide_Cas13bC- GACGTATGTACCAGGTTCCTGGAACTGGAATCTCTTTCCATAluc_84_ADAR27Top GAATGTTCTAAACCATCCTGCGGCCTCTACTCTGCATTCAATT(SEQ ID NO: 281) Guide_Cas13bC-GACACGTATGTACCAGGTTCCTGGAACTGGAATCTCTTTCCA luc_84_ADAR28TopTAGAATGTTCTAAACCATCCTGCGGCCTCTACTCTGCATTCAA (SEQ ID NO: 282)Guide_Cas13bC- GCAACACGTATGTACCAGGTTCCTGGAACTGGAATCTCTTTCluc_84_ADAR29Top CATAGAATGTTCTAAACCATCCTGCGGCCTCTACTCTGCATTC(SEQ ID NO: 283) Guide_Cas13bC-GCCCAACACGTATGTACCAGGTTCCTGGAACTGGAATCTCTT luc_84_ADAR30TopTCCATAGAATGTTCTAAACCATCCTGCGGCCTCTACTCTGCAT (SEQ ID NO: 284)Guide_Cas13bC- GGACCCAACACGTATGTACCAGGTTCCTGGAACTGGAATCTCluc_84_ADAR31Top TTTCCATAGAATGTTCTAAACCATCCTGCGGCCTCTACTCTGC(SEQ ID NO: 285) Guide_Cas13bC-GTTGACCCAACACGTATGTACCAGGTTCCTGGAACTGGAATC luc_84_ADAR32TopTCTTTCCATAGAATGTTCTAAACCATCCTGCGGCCTCTACTCT (SEQ ID NO: 286)Guide_Cas13bC- GCCTTGACCCAACACGTATGTACCAGGTTCCTGGAACTGGAAluc_84_ADAR33Top TCTCTTTCCATAGAATGTTCTAAACCATCCTGCGGCCTCTACT(SEQ ID NO: 287) Guide_Cas13bC-GTTCCTTGACCCAACACGTATGTACCAGGTTCCTGGAACTGG luc_84_ADAR34TopAATCTCTTTCCATAGAATGTTCTAAACCATCCTGCGGCCTCTA (SEQ ID NO: 288)Guide_Cas13bC- GGGTTCCTTGACCCAACACGTATGTACCAGGTTCCTGGAACTluc_84_ADAR35Top GGAATCTCTTTCCATAGAATGTTCTAAACCATCCTGCGGCCTC(SEQ ID NO: 289) Guide_Cas13bC-GTTGGTTCCTTGACCCAACACGTATGTACCAGGTTCCTGGAA luc_84_ADAR36TopCTGGAATCTCTTTCCATAGAATGTTCTAAACCATCCTGCGGCC (SEQ ID NO: 290)Guide_Cas13bC- GCCTTGGTTCCTTGACCCAACACGTATGTACCAGGTTCCTGGluc_84_ADAR37Top AACTGGAATCTCTTTCCATAGAATGTTCTAAACCATCCTGCGG(SEQ ID NO: 291 Guide_Cas13bC-GGCCCTTGGTTCCTTGACCCAACACGTATGTACCAGGTTCCT luc_84_ADAR38TopGGAACTGGAATCTCTTTCCATAGAATGTTCTAAACCATCCTGC (SEQ ID NO: 292)Guide_Cas13bC- GCCGCCCTTGGTTCCTTGACCCAACACGTATGTACCAGGTTCluc_84_ADAR39Top CTGGAACTGGAATCTCTTTCCATAGAATGTTCTAAACCATCCT(SEQ ID NO: 293) Guide_Cas13bC-GCGCCGCCCTTGGTTCCTTGACCCAACACGTATGTACCAGGT luc_84_ADAR40TopTCCTGGAACTGGAATCTCTTTCCATAGAATGTTCTAAACCATC (SEQ ID NO: 294)Guide_Cas13bC- GGTCGCCGCCCTTGGTTCCTTGACCCAACACGTATGTACCAGluc_84_ADAR41Top GTTCCTGGAACTGGAATCTCTTTCCATAGAATGTTCTAAACCA(SEQ ID NO: 295)

Reversing Causal Disease Mutations

The three genes in the Table below are synthesised with the pathogenicG>A mutation that introduces a pre-termination stop site into the geneand integrate them into a non-human cell line. The ability ofCas13b12-ADAR2 to correct the transcripts by changing the stop codon UAGto UIG (UGG) and thus restore protein translation is tested.

TABLE 11 Protein length (aa) Full length candidates Gene Disease 498NM_004992.3(MECP2): c.311G > A MECP2 Rett syndrome (p.Trp104Ter) 414NM_007375.3(TARDBP): c.943G > A TARDBP Amyotrophic lateral (p.Ala315Thr)sclerosis type 10 393 NM_000546.5(TP53): c.273G > A TP53 Li-Fraumeni(p.Trp91Ter) syndrome|Hereditary cancer-predisposing syndrome

Forty-eight more pathogenic G>A mutations are shown in Table 5 belowalong with the accompanying disease. 200 bp fragments around thesemutations are synthesised, rather than the entire gene, and cloned infront of a GFP. When the pre-termination site is restored, that willallow translation of the GFP and correction can be measured byfluorescence in high-throughput—in addition to RNA sequencing.

TABLE 12 Candidate Gene Disease 1 NM_004006.2(DMD): c.3747G > A DMDDuchenne muscular (p.Trp1249Ter) dystrophy 2 M_000344.3(SMNI): c.305G >A SMN1 Spinal muscular atrophy, (p.Trp102Ter) type II|Kugelberg-Welander disease 3 NM_000492.3(CFTR): c.3846G > A CFTR Cysticfibrosis|Hereditary (p.Trp1282Ter) pancreatitis|not provided|atalurenresponse - Efficacy 4 NM_004562.2(PRKN): c.1358G > A PRKN Parkinsondisease 2 (p.Trp453Ter) 5 NM_017651.4(AHI1): c.2174G > A AHI1 Joubertsyndrome 3 (p.Trp725Ter) 6 NM_000238.3(KCNH2): c.3002G > A KCNH2 Long QTsyndrome|not (p.Trp1001Ter) provided 7 NM_000136.2(FANCC): c.1517G > AFANCC|C9orf3 Fanconi anemia, (p.Trp506Ter) complementation group C 8NM_001009944.2(PKD1): c.12420G > A PKD1 Polycystic kidney disease,(p.Trp4140Ter) adult type 9 NM_177965.3(C8orf37): c.555G > A C8orf37Retinitis pigmentosa 64 (p.Trp185Ter) 10 NM_000833.4(GRIN2A): c.3813G >A GRIN2A Epilepsy, focal, with (p.Trp1271Ter) speech disorder and withor without mental retardation 11 NM_000548.4(TSC2): c.2108G > A TSC2Tuberous sclerosis (pTrp703Ter) 2|Tuberous sclerosis syndrome 12NM_000267.3(NF1): c.7044G > A NF1 Neurofibromatosis, type 1(p.Trp2348Ter) 13 NM_000520.5(HEXA): c.1454G > A HEXA Tay-Sachs disease(p.Trp485Ter) 14 NM_130838.1(UBE3A): c.2304G > A UBE3A Angelman syndrome(p.Trp768Ter) 15 NM_000543.4(SMPD1): c.168G A SMPD1 Niemann-Pickdisease, (pTrp56Ter) type A 16 NM_000218.2(KCNQ1): c.1175G > A KCNQ1Long QT syndrome (p.Trp392Ter) 17 NM_000256.3(MYBPC3): c.3293G > AMYBPC3 Primary familial (p.Trp1098Ter) hypertrophic cardiomyopathy 18(NM_000038.5(APC): c.1262G > A APC Familial adenomatous (p.Trp421Ter)polyposis 1 19 NM_000249.3(MLH1): c.1998G > A MLH1 Lynch syndrome(p.Trp666Ter) 20 NM_000054.4(AVPR2): c.878G > A AVPR2 Nephrogenicdiabetes (p.Trp293Ter) insipidus, X-linked 21 NM_001204.6(BMPR2):c.893G > A BMPR2 Primary pulmonary (p.W298*) hypertension 22NM_004560.3(ROR2): c.2247G > A ROR2 Brachydactyly type B1 (p.Trp749Ter)23 NM_000518.4(HBB): c.114G > A HBB beta{circumflex over( )}0{circumflex over ( )} Thalassemia|beta (p.Trp38Ter) Thalassemia 24NM_024577.3(SH3TC2): c.920G > A SH3TC2 Charcot-Marie-Tooth (p.Trp307Ter)disease, type 4C 25 NM_206933.2(USH2A): c.9390G > A USH2A Ushersyndrome, type 2A (p.Trp3130Ter) 26 NM_000179.2(MSH6): c.3020G > A MSH6Lynch syndrome (p.Trp1007Ter) 27 NM_002977.3(SCN9A): c.2691G > ASCN9A|LOC101929680 Indifference to pain, (p.Trp897Ter) congenital,autosomal recessive 28 NM_000090.3(COL3A1): c.30G > A COL3A1Ehlers-Danlos syndrome, (p.Trp10Ter) type 4 29 NM_000551.3(VHL):c.263G > A VHL Von Hippel-Lindau (p.Trp88Ter) syndrome|not provided 30NM_015627.2(LDLRAP1): c.65G > A LDLRAP1 Hypercholesterolemia,(p.Trp22Ter) autosomal recessive 31 NM_000132.3(F8): c.3144G > A F8Hereditary factor VIII (p.Trp1048Ter) deficiency disease 32NM_002185.4(IL7R): c.651G > A IL7R Severe combined (p.Trp217Ter)immunodeficiency, autosomal recessive, T cell-negative, B cell-positive, NK cell-positive 33 NM_000527.4(LDLR): c.1449G > A LDLRFamilial (p.Trp483Ter) hypercholesterolemia 34 NM_002294.2(LAMP2):c.962G > A LAMP2 Danon disease (p.Trp321Ter) 35 NM_000271.4(NPC1):c.1142G > A NPC1 Niemann-Pick disease (p.Trp381Ter) type C1 36NM_000267.3(NF1): c.1713G > A NF1 Neurofibromatosis, type 1(p.Trp571Ter) 37 NM_000035.3(ALDOB): c.888G > A ALDOB Hereditaryfructosuria (p.Trp296Ter) 38 NM_000090.3(COL3A1): c.3833G > A COL3A1Ehlers-Danlos syndrome, (p.Trp1278Ter) type 4 39 NM_001369.2(DNAH5):c.8465G > A DNAH5 Primary ciliary dyskinesia (p.Trp2822Ter) 40NM_178443.2(FERMT3): c.48G > A FERMT3 Leukocyte adhesion (p.Trp16Ter)deficiency, type III 41 NM_005359.5(SMAD4): c.906G > A SMAD4 Juvenilepolyposis (p.Trp302Ter) syndrome 42 NM_032119.3(ADGRV1): c.7406G > AADGRV1 Usher syndrome, type 2C (p.Trp2469Ter) 43 NM_000206.2(IL2RG):c.710G > A IL2RG X-linked severe combined (p.Trp237Ter) immunodeficiency44 NM_007294.3(BRCA1): c.5511G > A BRCA1 Familial cancer of(p.Trp1837Ter) breast|Breast-ovarian cancer, familial 1 45NM_130799.2(MEN1): c.1269G > A MEN1 Hereditary cancer- (p.Trp423Ter)predisposing syndrome 46 NM_000071.2(CBS): c.162G > A CBS Homocystinuriadue to (p.Trp54Ter) CBS deficiency 47 NM_000059.3(BRCA2): c.582G > ABRCA2 Familial cancer of (p.Trp194Ter) breast|Breast-ovarian cancer,familial 2 48 NM_000053.3(ATP7B): c.2336G > A ATP7B Wilson disease(p.Trp779Ter)

Example 3

Efficient and precise nucleic acid editing holds great promise fortreating genetic disease, particularly at the level of RNA, wheredisease-relevant transcripts can be rescued to yield functional proteinproducts. Type VI CRISPR-Cas systems contain the programmablesingle-effector RNA-guided RNases Cas13. Here, we profile the diversityof Type VI systems to engineer a Cas13 ortholog capable of robustknockdown and demonstrate RNA editing by using catalytically-inactiveCas13 (dCas13) to direct adenosine deaminase activity to transcripts inmammalian cells. By fusing the ADAR2 deaminase domain to dCas13 andengineering guide RNAs to create an optimal RNA duplex substrate, weachieve targeted editing of specific single adenosines to inosines(which is read out as guanosine during translation) with efficienciesroutinely ranging from 20-40% and up to 89%. This system, referred to asRNA Editing for Programmable A to I Replacement (REPAIR), can be furtherengineered to achieve high specificity. An engineered variant, REPAIRv2,displays greater than 170-fold increase in specificity while maintainingrobust on-target A to I editing. We use REPAIRv2 to edit full-lengthtranscripts containing known pathogenic mutations and create functionaltruncated versions suitable for packaging in adeno-associated viral(AAV) vectors. REPAIR presents a promising RNA editing platform withbroad applicability for research, therapeutics, and biotechnology.Precise nucleic acid editing technologies are valuable for studyingcellular function and as novel therapeutics. Although current editingtools, such as the Cas9 nuclease, can achieve programmable modificationof genomic loci, edits are often heterogenous due to insertions ordeletions or require a donor template for precise editing. Base editors,such as dCas9-APOBEC fusions, allow for editing without generating adouble stranded break, but may lack precision due to the nature ofcytidine deaminase activity, which edits any cytidine in a targetwindow. Furthermore, the requirement for a protospacer adjacent motif(PAM) limits the number of possible editing sites. Here, we describe thedevelopment of a precise and flexible RNA base editing tool using theRNA-guided RNA targeting Cas13 enzyme from type VI prokaryotic clusteredregularly interspaced short palindromic repeats (CRISPR) adaptive immunesystem.

Precise nucleic acid editing technologies are valuable for studyingcellular function and as novel therapeutics. Current editing tools,based on programmable nucleases such as the prokaryotic clusteredregularly interspaced short palindromic repeats (CRISPR)-associatednucleases Cas9 (1-4) or Cpf1(5), have been widely adopted for mediatingtargeted DNA cleavage which in turn drives targeted gene disruptionthrough non-homologous end joining (NHEJ) or precise gene editingthrough template-dependent homology-directed repair (HDR)(6). NHEJutilizes host machineries that are active in both dividing andpost-mitotic cells and provides efficient gene disruption by generatinga mixture of insertion or deletion (indel) mutations that can lead toframe shifts in protein coding genes. HDR, in contrast, is mediated byhost machineries whose expression is largely limited to replicatingcells. As such, the development of gene-editing capabilities inpost-mitotic cells remains a major challenge. Recently, DNA baseeditors, such as the use of catalytically inactive Cas9 (dCas9) totarget cytidine deaminase activity to specific genome targets to effectcytosine to thymine conversions within a target window, allow forediting without generating a DNA double strand break and significantlyreduces the formation of indels(7, 8). However the targeting range ofDNA base editors is limited due to the requirement of Cas9 for aprotospacer adjacent motif (PAM) at the editing site(9). Here, wedescribe the development of a precise and flexible RNA base editingtechnology using the type VI CRISPR-associated RNA-guided RNaseCas13(10-13).

Cas13 enzymes have two Higher Eukaryotes and ProkaryotesNucleotide-binding (HEPN) endoRNase domains that mediate precise RNAcleavage(10, 11). Three Cas13 protein families have been identified todate: Cas13a (previously known as C2c2), Cas13b, and Cas13c(12, 13). Werecently reported Cas13a enzymes can be adapted as tools for nucleicacid detection(14) as well as mammalian and plant cell RNA knockdown andtranscript tracking(15). The RNA-guided nature of Cas13 enzymes makesthem attractive tool for RNA binding and perturbation applications.

The adenosine deaminase acting on RNA (ADAR) family of enzymes mediatesendogenous editing of transcripts via hydrolytic deamination ofadenosine to inosine, a nucleobase that is functionally equivalent toguanosine in translation and splicing(16). There are two functionalhuman ADAR orthologs, ADAR1 and ADAR2, which consist of N-terminaldouble stranded RNA-binding domains and a C-terminal catalyticdeamination domain. Endogenous target sites of ADAR1 and ADAR2 containsubstantial double stranded identity, and the catalytic domains requireduplexed regions for efficient editing in vitro and in vivo(17, 18).Although ADAR proteins have preferred motifs for editing that couldrestrict the potential flexibility of targeting, hyperactive mutants,such as ADAR(E488Q)(19), relax sequence constraints and improveadenosine to inosine editing rates. ADARs preferentially deaminateadenosines opposite cytidine bases in RNA duplexes(20), providing apromising opportunity for precise base editing. Although previousapproaches have engineered targeted ADAR fusions via RNA guides (21-24),the specificity of these approaches has not been reported and theirrespective targeting mechanisms rely on RNA-RNA hybridization withoutthe assistance of protein partners that may enhance target recognitionand stringency.

Here we assay the entire family of Cas13 enzymes for RNA knockdownactivity in mammalian cells and identify the Cas13b ortholog fromPrevotella sp. P5-125 (PspCas13b) as the most efficient and specific formammalian cell applications. We then fuse the ADAR2 deaminase domain(ADARDD) to catalytically inactive PspCas13b and demonstrate RNA editingfor programmable A to I (G) replacement (REPAIR) of reporter andendogenous transcripts as well as disease-relevant mutations. Lastly, weemploy a rational mutagenesis scheme to improve the specificity ofdCas13b-ADAR2DD fusions to generate REPAIRv2 with more than 170 foldincrease in specificity.

Methods Design and Cloning of Bacterial Constructs

Mammalian codon optimized Cas13b constructs were cloned into thechloramphenicol resistant pACYC184 vector under control of the Lacpromoter. Two corresponding direct-repeat (DR) sequences separated byBsaI restriction sites were then inserted downstream of Cas13b, undercontrol of the pJ23119 promoter. Last, oligos for targeting spacers werephosphorylated using T4 PNK (New England Biolabs), annealed and ligatedinto BsaI digested vectors using T7 ligase (Enzymatics) to generatetargeting Cas13b vectors.

Bacterial PFS Screens

Ampicillin resistance plasmids for PFS screens were cloned by insertingPCR products containing Cas13b targets with 2 5′ randomized nucleotidesand 4 3′ randomized nucleotides separated by a target site immediatelydownstream of the start codon of the ampicillin resistance gene blausing NEB Gibson Assembly (New England Biolabs). 100 ng ofampicillin-resistant target plasmids were then electroporated with65-100 ng chloramphenicol-resistant Cas13b bacterial targeting plasmidsinto Endura Electrocompetent Cells. Plasmids were added to cells,incubated 15 minutes on ice, electroporated using the manufacturer'sprotocol, and then 950 uL of recovery media was added to cells before aone hour outgrowth at 37 C. The outgrowth was plated ontochloramphenicol and ampicillin double selection plates. Serial dilutionsof the outgrowth were used to estimate the cfu/ng DNA. 16 hours postplating, cells were scraped off plates and surviving plasmid DNAharvested using the Qiagen Plasmid Plus Maxi Kit (Qiagen). SurvivingCas13b target sequences and their flanking regions were amplified by PCRand sequenced using an Illumina NextSeq. To assess PFS preferences, thepositions containing randomized nucleotides in the original library wereextracted, and sequences depleted relative to the vector only conditionthat were present in both bioreplicates were extracted using custompython scripts. The −log2 of the ratio of PFS abundance in the Cas13bcondition compared to the vector only control was then used to calculatepreferred motifs. Specifically, all sequences having−log2(sample/vector) depletion ratios above a specific threshold wereused to generate weblogos of sequence motifs (weblogo.berkeley.edu). Thespecific depletion ratio values used to generate weblogos for eachCas13b ortholog are listed in Table 9.

Design and Cloning of Mammalian Constructs for RNA Interference

To generate vectors for testing Cas13 orthologs in mammalian cells,mammalian codon optimized Cas13a, Cas13b, and Cas13c genes were PCRamplified and golden-gate cloned into a mammalian expression vectorcontaining dual NLS sequences and a C-terminal msfGFP, under control ofthe EF1alpha promoter. For further optimization Cas13 orthologs weregolden gate cloned into destination vectors containing differentC-terminal localization tags under control of the EF1alpha promoter.

The dual luciferase reporter was cloned by PCR amplifying Gaussia andCypridinia luciferase coding DNA, the EF1alpha and CMV promoters andassembly using the NEB Gibson Assembly (New England Biolabs).

For expression of mammalian guide RNA for Cas13a, Cas13b, or Cas13corthologs, the corresponding direct repeat sequences were synthesizedwith golden-gate acceptor sites and cloned under U6 expression viarestriction digest cloning. Individual guides were then cloned into thecorresponding expression backbones for each ortholog by golden gatecloning.

Cloning of Pooled Mismatch Libraries for Cas13 Interference Specificity

Pooled mismatch library target sites were created by PCR. Oligoscontaining semi-degenerate target sequences in G-luciferase containing amixture of 94% of the correct base and 2% of each incorrect base at eachposition within the target were used as one primer, and an oligocorresponding to a non-targeted region of G-luciferase was used as thesecond primer in the PCR reaction. The mismatch library target was thencloned into the dual luciferase reporter in place of the wildtypeG-luciferase using NEB Gibson assembly (New England Biolabs).

Design and Cloning of Mammalian Constructs for RNA Editing

PspCas13b was made catalytically inactive (dPspCas13b) via two histidineto alanine mutations (H133A/H1058A) at the catalytic site of the HEPNdomains. The deaminase domains of human ADAR1 and ADAR2 were synthesizedand PCR amplified for gibson cloning into pcDNA-CMV vector backbones andwere fused to dPspCas13b at the C-terminus via GS or GSGGGGS (SEQ ID NO:296) linkers. For the experiment in which we tested different linkers wecloned the following additional linkers between dPspCas13b and ADAR2dd:GGGGSGGGGSGGGGS, EAAAK (SEQ ID NO: 297) , GGSGGSGGSGGSGGSGGS (SEQ ID NO:298), and SGSETPGTSESATPES (XTEN) (SEQ ID NO: 299). Specificity mutantswere generated by gibson cloning the appropriate mutants into thedPspCas13b-GSGGGGS (SEQ ID NO: 761) backbone.

The luciferase reporter vector for measuring RNA editing activity wasgenerated by creating a W85X mutation (TGG>TAG) in the luciferasereporter vector used for knockdown experiments. This reporter vectorexpresses functional Gluc as a normalization control, but a defectiveCluc due to the addition of a pretermination site. To test ADAR editingmotif preferences, we cloned every possible motif around the adenosineat codon 85 (XAX) of Cluc.

For testing PFS preference of REPAIR, we cloned a pooled plasmid librarycontaining a 6 basepair degenerate PFS sequence upstream of a targetregion and adenosine editing site. The library was synthesized as anultramer from Integrated DNA Technologies (IDT) and was made doublestranded via annealing a primer and Klenow fragment of DNA polymerase I(New England Biolabs) fill in of the sequence. This dsDNA fragmentcontaining the degenerate sequence was then gibson cloned into thedigested reporter vector and this was then isopropanol precipitated andpurified. The cloned library was then electroporated into Enduracompetent E. coli cells (Lucigen) and plated on 245 mm×245 mm squarebioassay plates (Nunc). After 16 hours, colonies were harvested andmidiprepped using endotoxin-free MACHEREY-NAGEL midiprep kits. Clonedlibraries were verified by next generation sequencing.

For cloning disease-relevant mutations for testing REPAIR activity, 34G>A mutations related to disease pathogenesis as defined in ClinVar wereselected and 200 bp regions surrounding these mutations were golden gatecloned between mScarlett and EGFP under a CMV promoter. Two additionalG>A mutations in AVPR2 and FANCC were selected for Gibson cloning thewhole gene sequence under expression of EF1alpha.

For expression of mammalian guide RNA for REPAIR, the PspCas13b directrepeat sequences were synthesized with golden-gate acceptor sites andcloned under U6 expression via restriction digest cloning. Individualguides were then cloned into this expression backbones by golden gatecloning.

Mammalian Cell Culture

Mammalian cell culture experiments were performed in the HEK293FT line(American Type Culture Collection (ATCC)), which was grown in Dulbecco'sModified Eagle Medium with high glucose, sodium pyruvate, and GlutaMAX(Thermo Fisher Scientific), additionally supplemented with 1×penicillin-streptomycin (Thermo Fisher Scientific) and 10% fetal bovineserum (VWR Seradigm). Cells were maintained at confluency below 80%.

Unless otherwise noted, all transfections were performed withLipofectamine 2000 (Thermo Fisher Scientific) in 96-well plates coatedwith poly-D-lysine (BD Biocoat). Cells were plated at approximately20,000 cells/well sixteen hours prior to transfection to ensure 90%confluency at the time of transfection. For each well on the plate,transfection plasmids were combined with Opti-MEM I Reduced Serum Medium(Thermo Fisher) to a total of 25 μl. Separately, 24.5 ul of Opti-MEM wascombined with 0.5 ul of Lipofectamine 2000. Plasmid and Lipofectaminesolutions were then combined and incubated for 5 minutes, after whichthey were pipetted onto cells.

RNA Knockdown Mammalian Cell Assays

To assess RNA targeting in mammalian cells with reporter constructs, 150ng of Cas13 construct was co-transfected with 300 ng of guide expressionplasmid and 12.5 ng of the knockdown reporter construct. 48 hourspost-transfection, media containing secreted luciferase was removed fromcells, diluted 1:5 in PBS, and measured for activity with BioLuxCypridinia and Biolux Gaussia luciferase assay kits (New EnglandBiolabs) on a plate reader (Biotek Synergy Neo2) with an injectionprotocol. All replicates performed are biological replicates.

For targeting of endogenous genes, 150 ng of Cas13 construct wasco-transfected with 300 ng of guide expression plasmid. 48 hourspost-transfection, cells were lysed and RNA was harvested and reversetranscribed using a previously described [CITE PROTOCOLS] modificationof the Cells-to-Ct kit (Thermo Fisher Scientific). cDNA expression wasmeasured via qPCR using TaqMan qPCR probes for the KRAS transcript(Thermo Fisher Scientific), GAPDH control probes (Thermo FisherScientific), and Fast Advanced Master Mix (Thermo Fisher Scientific).qPCR reactions were read out on a LightCycler 480 Instrument II (Roche),with four 5 ul technical replicates in 384-well format.

Evaluation of RNA Specificity Using Pooled Library of Mismatched Targets

The ability of Cas13 to interfere with the mismatched target library wastested using HEK293FT cells seeded in 6 well plates. ˜70% confluentcells were transfected using 2400 ng Cas13 vector, 4800 ng of guide and240 ng of mismatched target library. 48 hours post transfection, cellswere harvested and RNA extracted using the QIAshredder (Qiagen) and theQiagen RNeasy Mini Kit. lug of extracted RNA was reverse transcribedusing the qScript Flex cDNA synthesis kit (Quantabio) following themanufacturer's gene-specific priming protocol and a Gluc specific RTprimer. cDNA was then amplified and sequenced on an Illumina NextSeq.

The sequencing was analyzed by counting reads per sequence and depletionscores were calculated by determining the log2(-read count ratio) value,where read count ratio is the ratio of read counts in the targetingguide condition versus the non-targeting guide condition. This scorevalue represents the level of Cas13 activity on the sequence, withhigher values representing stronger depletion and thus higher Cas13cleavage activity. Separate distributions for the single mismatch anddouble mismatch sequences were determined and plotted as heatmaps with adepletion score for each mismatch identity. For double mismatchsequences the average of all possible double mismatches at a givenposition were plotted.

Transcriptome-Wide Profiling of Cas13 in Mammalian Cells by RNASequencing

For measurement of transcriptome-wide specificity, 150 ng of Cas13construct, 300 ng of guide expression plasmid and 15 ng of the knockdownreporter construct were co-transfected; for shRNA conditions, 300 ng ofshRNA targeting plasmid, 15 ng of the knockdown reporter construct, and150 ng of EF1-alpha driven mCherry (to balance reporter load) wereco-transfected. 48 hours after transfection, RNA was purified with theRNeasy Plus Mini kit (Qiagen), mRNA was selected for using NEBNextPoly(A) mRNA Magnetic Isolation Module (New England Biolabs) andprepared for sequencing with the NEBNext Ultra RNA Library Prep Kit forIllumina (New England Biolabs). RNA sequencing libraries were thensequenced on a NextSeq (Illumina).

To analyze transcriptome-wide sequencing data, reads were aligned RefSeqGRCh38 assembly using Bowtie and RSEM version 1.2.31 with defaultparameters [CITE RSEM: accurate transcript quantification from RNA-Seqdata with or without a reference genome]. Transcript expression wasquantified as log2(TPM+1), genes were filtered for log2(TPM+1) >2.5 Forselection of differentially expressed genes, only genes withdifferential changes of >2 or <0.75 were considered. Statisticalsignificance of differential expression was evaluated Student's T-teston three targeting replicates versus non-targeting replicates, andfiltered for a false discovery rate of <0.01% by Benj amini-Hochb ergprocedure.

ADAR RNA Editing in Mammalian Cells Transfections

To assess REPAIR activity in mammalian cells, we transfected 150 ng ofREPAIR vector, 300 ng of guide expression plasmid, and 40 ng of the RNAediting reporter. After 48 hours, RNA from cells were harvested andreverse transcribed using a method previously described [cite JJ] with agene specific reverse transcription primer. The extracted cDNA was thensubjected to two rounds of PCR to add Illumina adaptors and samplebarcodes using NEBNext High-Fidelity 2X PCR Master Mix. The library wasthen subjected to next generation sequencing on an Illumina NextSeq orMiSeq. RNA editing rates were then evaluated at all adenosine within thesequencing window.

In experiments where the luciferase reporter was targeted for RNAediting, we also harvested the media with secreted luciferase prior toRNA harvest. In this case, because the corrected Cluc might be at lowlevels, we did not dilute the media. We measured luciferase activitywith BioLux Cypridinia and Biolux Gaussia luciferase assay kits (NewEngland Biolabs) on a plate reader (Biotek Synergy Neo2) with aninjection protocol. All replicates performed are biological replicates.

PFS Binding Mammalian Screen

To determine the contribution of the PFS to editing efficiency, 625 ngof PFS target library, 4.7 ug of guide, and 2.35 ug of REPAIR wereco-transfected on HEK293FT cells plated in 225 cm2 flasks. Plasmids weremixed with 33 ul of PLUS reagent (Thermo Fisher Scientific), brought to533 ul with Opti-MEM, incubated for 5 minutes, combined with 30 ul ofLipofectamine 2000 and 500 ul of Opti-MEM, incubated for an additional 5minutes, and then pipetted onto cells. 48 hours post-transfection, RNAwas harvested with the RNeasy Plus Mini kit (Qiagen), reversetranscribed with qScript Flex (Quantabio) using a gene specific primer,and amplified with two rounds of PCR using NEBNext High-Fidelity 2× PCRMaster Mix (New England Biolabs) to add Illumina adaptors and samplebarcodes. The library was sequenced on an Illumina NextSeq, and RNAediting rates at the target adenosine were mapped to PFS identity. Toincrease coverage, the PFS was computationally collapsed to 4nucleotides. REPAIR editing rates were calculated for each PFS, averagedover biological replicates with non-targeting rates for thecorresponding PFS subtracted.

Whole-Transcriptome Sequencing to Evaluate ADAR Editing Specificity

For analyzing off-target RNA editing sites across the transcriptome, weharvested total RNA from cells 48 hours post transfection using theRNeasy Plus Miniprep kit (Qiagen). The mRNA fraction is then enrichedusing a NEBNext Poly(A) mRNA Magnetic Isolation Module (NEB) and thisRNA is then prepared for sequencing using NEBNext Ultra RNA Library PrepKit for Illumina (NEB). The libraries were then sequenced on an IlluminaNextSeq and loaded such that there was at least 5 million reads persample.

RNA Editing Analysis for Targeted and Transcriptome Wide Experiments

To analyze the transcriptome-wide RNA editing RNA sequencing data,sequence files were randomly downsampled to 5 million reads. An indexwas generated using the RefSeq GRCh38 assembly with Gluc and Clucsequences added and reads were aligned and quantified using Bowtie/RSEMversion 1.3.0. Alignment BAMs were then sorted and analyzed for RNAediting sites using REDitools [cite] with the following parameters: -t 8-e -d -1 -U [AG or TC] -p -u -m20 -T6-0 -W -v 1 -n 0.0. Any significantedits found in untransfected or EGFP-transfected conditions wereconsidered to be SNPs or artifacts of the transfection and filtered outfrom the analysis of off-targets. Off-targets were consideredsignificant if the Fisher's exact test yielded a p-value less than 0.5and that at least 2 of 3 biological replicates identified the edit site.

For analyzing the predicted variant effects of each off-target, the listof off-target edit sites was analyzed using the variant annotationintegrator (genome.ucsc.edu/cgi-bin/hgVai) as part of the UCSC genomebrowser suite of tools using the SIFT and PolyPhen-2 annotations. Todeclare whether the off-target genes are oncogenic, a database ofoncogenic annotations from the COSMIC catalogue of somatic mutations incancer (cancer.sanger.ac.uk).

For analyzing whether the REPAIR constructs perturbed RNA levels, thetranscript per million (TPM) values output from the RSEM analysis wereused for expression counts and transformed to log-space by taking thelog2(TPM+1). To find differentially regulated genes, a Student's t-testwas performed on three targeting guide replicates versus threenon-targeting guide replicates. The statistical analysis was onlyperformed on genes with log2(TPM+1) values greater than 2.5 and geneswere only considered differentially regulated if they had a fold changegreater than 2 or less than 0.8. Genes were reported if they had a falsediscovery rate of less than 0.01.

Results Comprehensive Characterization of Cas13 Family Members inMammalian Cells

We previously developed LwaCas13a for mammalian knockdown applications,but it required an msfGFP stabilization domain for efficient knockdownand, although the specificity was high, knockdown efficiencies were notconsistently below 50%(15). We sought to identify a more robustRNA-targeting CRISPR system by characterizing a genetically diverse setof Cas13 family members to assess their RNA knockdown activity inmammalian cells (FIG. 49A). We cloned 21 Cas13a, 15 Cas13b, and 7 Cas13cmammalian codon-optimized orthologs (Table 6) into an expression vectorwith N- and C-terminal nuclear export signal (NES) sequences and aC-terminal msfGFP to enhance protein stability. To assay interference inmammalian cells, we designed a dual reporter construct expressing theorthogonal Gaussia (Gluc) and Cypridinia (Cluc) luciferases underseparate promoters, which allows one luciferase to function as a measureof Cas13 interference activity and the other to serve as an internalcontrol. For each ortholog, we designed PFS-compatible guide RNAs, usingthe Cas13b PFS motifs derived from an ampicillin interference assay(FIGS. 55A-55C; Table 7) and the 3′ H PFS from previous reports ofCas13a activity(10).

We transfected HEK293FT cells with Cas13 expression, guide RNA andreporter plasmids and quantified levels of the targeted Gluc 48 hourslater. Testing two guide RNAs for each Cas13 ortholog revealed a rangeof activity levels, including five Cas13b orthologs with similar orincreased interference across both guide RNAs relative to the recentlycharacterized LwaCas13a (FIG. 49B). We selected these five Cas13borthologs, as well as the top two Cas13a orthologs for furtherengineering.

We next tested for Cas13-mediated knockdown of Gluc without msfGFP, inorder to select orthologs that do not require stabilization domains forrobust activity. We hypothesized that, in addition to msfGFP, Cas13activity could be affected by subcellular localization, as previouslyreported for optimization of LwaCas13a(15). Therefore, we tested theinterference activity of the seven selected Cas13 orthologs C-terminallyfused to one of six different localization tags without msfGFP. Usingthe luciferase reporter assay, we found that PspCas13b and PguCas13bC-terminally fused to the HIV Rev gene NES and RanCas13b C-terminallyfused to the MAPK NES had the highest levels of interference activity(FIG. 56A). To further distinguish activity levels of the top orthologs,we compared the three optimized Cas13b constructs to the optimalLwaCas13a-msfGFP fusion and shRNA for their ability to knockdown theKRAS transcript using position-matched guides (FIG. 56B). We observedthe highest levels interference for PspCas13b (average knockdown 62.9%)and thus selected this for further comparison to LwaCas13 a.

To more rigorously define the activity level of PspCas13b and LwaCas13awe designed position matched guides tiling along both Gluc and Cluc andassayed their activity using our luciferase reporter assay. We tested 93and 20 position matched guides targeting Gluc and Cluc, respectively,and found that PspCas13b had consistently increased levels of knockdownrelative to LwaCas13a (average of 92.3% for PspCas13b vs. 40.1%knockdown for LwaCas13a) (FIGS. 49C, 49D).

Specificity of Cas13 Mammalian Interference Activity

To characterize the interference specificities of PspCas13b andLwaCas13a we designed a plasmid library of luciferase targets containingsingle mismatches and double mismatches throughout the target sequenceand the three flanking 5′ and 3′ base pairs (FIG. 56C). We transfectedHEK293FT cells with either LwaCas13a or PspCas13b, a fixed guide RNAtargeting the unmodified target sequence, and the mismatched targetlibrary corresponding to the appropriate system. We then performedtargeted RNA sequencing of uncleaved transcripts to quantify depletionof mismatched target sequences. We found that LwaCas13a and PspCas13bhad a central region that was relatively intolerant to singlemismatches, extending from base pairs 12-26 for the PspCas13b target and13-24 for the LwaCas13a target (FIG. 56D). Double mismatches were evenless tolerated than single mutations, with little knockdown activityobserved over a larger window, extending from base pairs 12-29 forPspCas13b and 8-27 for LwaCas13a in their respective targets (FIG. 56E).Additionally, because there are mismatches included in the threenucleotides flanking the 5′ and 3′ ends of the target sequence, we couldassess PFS constraints on Cas13 knockdown activity. Sequencing showedthat almost all PFS combinations allowed robust knockdown, indicatingthat a PFS constraint for interference in mammalian cells likely doesnot exist for either enzyme tested. These results indicate that Cas13aand Cas13b display similar sequence constraints and sensitivitiesagainst mismatches.

We next characterized the interference specificity of PspCas13b andLwaCas13a across the mRNA fraction of the transcriptome. We performedtranscriptome-wide mRNA sequencing to detect significant differentiallyexpressed genes. LwaCas13a and PspCas13b demonstrated robust knockdownof Gluc (FIGS. 49E, 49F) and were highly specific compared to aposition-matched shRNA, which showed hundreds of off-targets (FIG. 49G).

Cas13-ADAR Fusions Enable Targeted RNA Editing

Given that PspCas13b achieved consistent, robust, and specific knockdownof mRNA in mammalian cells, we envisioned that it could be adapted as anRNA binding platform to recruit the deaminase domain of ADARs(ADAR_(DD)) for programmable RNA editing. To engineer a PspCas13blacking nuclease activity (dPspCas13b, referred to as dCas13b fromhere), we mutated conserved catalytic residues in the HEPN domains andobserved loss of luciferase RNA knockdown activity (FIG. 57A). Wehypothesized that a dCas13b-ADAR_(DD) fusion could be recruited by aguide RNA to target adenosines, with the hybridized RNA creating therequired duplex substrate for ADAR activity (FIG. 50A). To enhancetarget adenosine deamination rates we introduced two additionalmodifications to our initial RNA editing design: we introduced amismatched cytidine opposite the target adenosine, which has beenpreviously reported to increase deamination frequency, and fused dCas13bwith the deaminase domains of human ADAR1 or ADAR2 containinghyperactivating mutations to enhance catalytic activity(ADAR1_(DD)(E1008Q)(25) or ADAR2_(DD)(E488Q)(19)).

To test the activity of dCas13b-ADARDD we generated an RNA-editingreporter on Cluc by introducing a nonsense mutation (W85X (UGG→UAG)),which could functionally be repaired to the wildtype codon through A→Iediting (FIG. 50B) and then be detected as restoration of Clucluminescence. We evenly tiled guides with spacers 30, 50, 70 or 84nucleotides in length across the target adenosine to determine theoptimal guide placement and design (FIG. 50C). We found thatdCas13b-ADAR1DD required longer guides to repair the Cluc reporter,while dCas13b-ADAR2_(DD) was functional with all guide lengths tested(FIG. 50C). We also found that the hyperactive E488Q mutation improvedediting efficiency, as luciferase restoration with the wildtypeADAR2_(DD) was reduced (FIG. 57B). From this demonstration of activity,we chose dCas13b-ADAR2_(DD)(E488Q) for further characterization anddesignated this approach as RNA Editing for Programmable A to IReplacement version 1 (REPAIRv1).

To validate that restoration of luciferase activity was due to bona fideediting events, we measured editing of Cluc transcripts subject toREPAIRv1 directly via reverse transcription and targeted next-generationsequencing. We tested 30- and 50-nt spacers around the target site andfound that both guide lengths resulted in the expected A to I edit, with50-nt spacers achieving higher editing percentages (FIG. 50D,E, FIG.57C). We also observed that 50-nt spacers had an increased propensityfor editing at non-targeted adenosines, likely due to increased regionsof duplex RNA (FIG. 50E, FIG. 57C).

We next targeted an endogenous gene, PPIB. We designed 50-nt spacerstiling PPIB and found that we could edit the PPIB transcript with up to28% editing efficiency (FIG. 57D). To test if REPAIR could be furtheroptimized, we modified the linker between dCas13b and ADAR2_(DD)(E488Q)(FIG. 57E, Table 8) and found that linker choice modestly affectedluciferase activity restoration.

Defining the Sequence Parameters for RNA Editing

Given that we could achieve precise RNA editing at a test site, wewanted to characterize the sequence constraints for programming thesystem against any RNA target in the transcriptome. Sequence constraintscould arise from dCas13b targeting limitations, such as the PFS, or fromADAR sequence preferences(26). To investigate PFS constraints onREPAIRv1, we designed a plasmid library carrying a series of fourrandomized nucleotides at the 5′ end of a target site on the Cluctranscript (FIG. 51A). We targeted the center adenosine within either aUAG or AAC motif and found that for both motifs, all PFSs demonstrateddetectable levels of RNA editing, with a majority of the PFSs havinggreater than 50% editing at the target site (FIG. 51B). Next, we soughtto determine if the ADAR2_(DD) in REPAIRv1 had any sequence constraintsimmediately flanking the targeted base, as has been reported previouslyfor ADAR2_(DD)(26).We tested every possible combination of 5′ and 3′flanking nucleotides directly surrounding the target adenosine (FIG.51C), and found that REPAIRv1 was capable of editing all motifs (FIG.51D). Lastly, we analyzed whether the identity of the base opposite thetarget A in the spacer sequence affected editing efficiency and foundthat an A-C mismatch had the highest luciferase restoration with A-G,A-U, and A-A having drastically reduced REPAIRv1 activity (FIG. 57F).

Correction of Disease-Relevant Human Mutations Using REPAIRv1

To demonstrate the broad applicability of the REPAIRv1 system for RNAediting in mammalian cells, we designed REPAIRv1 guides against twodisease relevant mutations: 878G>A (AVPR2 W293X) in X-linked Nephrogenicdiabetes insipidus and 1517G>A (FANCC W506X) in Fanconi anemia. Wetransfected expression constructs for cDNA of genes carrying thesemutations into HEK293FT cells and tested whether REPAIRv1 could correctthe mutations. Using guide RNAs containing 50-nt spacers, we were ableto achieve 35% correction of AVPR2 and 23% correction of FANCC (FIG.52A-52D). We then tested the ability of REPAIRv1 to correct 34 differentdisease-relevant G>A mutations (Table 9) and found that we were able toachieve significant editing at 33 sites with up to 28% editingefficiency (FIG. 52E). The mutations we chose are only a fraction of thepathogenic G to A mutations (5,739) in the ClinVar database, which alsoincludes an additional 11,943 G to A variants (FIG. 52F and FIG. 58).Because there are no sequence constraints, REPAIRv1 is capable ofpotentially editing all these disease relevant mutations, especiallygiven that we observed significant editing regardless of the targetmotif (FIG. 51C and FIG. 52G).

Delivering the REPAIRv1 system to diseased cells is a prerequisite fortherapeutic use, and we therefore sought to design REPAIRv1 constructsthat could be packaged into therapeutically relevant viral vectors, suchas adeno-associated viral (AAV) vectors. AAV vectors have a packaginglimit of 4.7 kb, which cannot accommodate the large size ofdCas13b-ADAR_(DD) (4473 bp) along with promoter and expressionregulatory elements. To reduce the size, we tested a variety ofN-terminal and C-terminal truncations of dCas13 fused toADAR2_(DD)(E488Q) for RNA editing activity. We found that all C-terminaltruncations tested were still functional and able to restore luciferasesignal (FIG. 59), and the largest truncation, C-terminal Δ984-1090(total size of the fusion protein 4,152 bp) was small enough to fitwithin the packaging limit of AAV vectors.

Transcriptome-Wide Specificity of REPAIRv1

Although RNA knockdown with PspCas13b was highly specific, in ourluciferase tiling experiments, we observed off-target adenosine editingwithin the guide:target duplex (FIG. 50E). To see if this was awidespread phenomenon, we tiled an endogenous transcript, KRAS, andmeasured the degree of off-target editing near the target adenosine(FIG. 53A). We found that for KRAS, while the on-target editing rate was23%, there were many sites around the target site that also haddetectable A to G edits (FIG. 53B).

Because of the observed off-target editing within the guide:targetduplex, we evaluated all possible transcriptome off-targets byperforming RNA sequencing on all mRNAs. RNA sequencing revealed thatthere was a significant number A to G off-target events, with 1,732off-targets in the targeting condition and 925 off-targets in thenon-targeting condition, with 828 off-targets overlapping (FIGS. 53C,53D). Of all the editing sites across the transcriptome, the on-targetediting site had the highest editing rate, with 89% A to G conversion.

Given the high specificity of Cas13 targeting, we reasoned that theoff-targets may arise from ADAR. Two RNA-guided ADAR systems have beendescribed previously (FIG. 60A). The first utilizes a fusion ofADAR2_(DD) to the small viral protein lambda N (λN), which binds to theBoxB-λ RNA hairpin(22). A guide RNA with double BoxB-λ hairpins guidesADAR2_(DD) to edit sites encoded in the guide RNA(23). The second designutilizes full length ADAR2 (ADAR2) and a guide RNA with a hairpin thatthe double strand RNA binding domains (dsRBDs) of ADAR2 recognize(21,24). We analyzed the editing efficiency of these two systems compared toREPAIRv1 and found that the BoxB-ADAR2 and ADAR2 systems demonstrated63% and 36% editing rates, respectively, compared to the 89% editingrate achieved by REPAIRv1 (FIGS. 60B-60E). Additionally, the BoxB andADAR2 systems created 2018 and 174 observed off targets, respectively,in the targeting guide conditions, compared to the 1,229 off targets inthe REPAIRv1 targeting guide condition. Notably, all the conditions withthe two ADAR2DD-based systems (REPAIRv1 and BoxB) showed a highpercentage of overlap in their off-targets while the ADAR2 system had alargely distinct set of off-targets (FIG. 60F). The overlap inoff-targets between the targeting and non-targeting conditions andbetween REPAIRv1 and BoxB conditions suggest ADAR2_(DD) droveoff-targets independent of dCas13 targeting (FIG. 60F).

Improving Specificity of REPAIRv1 through Rational Protein Engineering

To improve the specificity of REPAIR, we employed structure-guidedprotein engineering of ADAR2_(DD)(E488Q). Because of theguide-independent nature of off-targets, we hypothesized thatdestabilizing ADAR2_(DD)(E488Q)-RNA binding would selectively decreaseoff-target editing, but maintain on-target editing due to increasedlocal concentration from dCas13b tethering of ADAR2_(DD)(E488Q) to thetarget site. We mutagenized ADAR2_(DD)(E488Q) residues previouslydetermined to contact the duplex region of the target RNA (FIG. 54A)(18)on the ADAR2_(DD)(E488Q) background. To assess efficiency andspecificity, we tested 17 single mutants with both targeting andnon-targeting guides, under the assumption that background luciferaserestoration in the non-targeting condition detected would be indicativeof broader off-target activity. We found that mutations at the selectedresidues had significant effects on the luciferase activity fortargeting and non-targeting guides (FIG. 54A, 54B, FIG. 61A). A majorityof mutants either significantly improved the luciferase activity for thetargeting guide or increased the ratio of targeting to non-targetingguide activity, which we termed the specificity score (FIG. 54A, 54B).We selected a subset of these mutants (FIG. 54B) for transcriptome-widespecificity profiling by next generation sequencing. As expected,off-targets measured from transcriptome-wide sequencing correlated withour specificity score (FIG. 61B) for mutants. We found that with theexception of ADAR2_(DD)(E488Q/R455E), all sequenced REPAIRv1 mutantscould effectively edit the reporter transcript (FIG. 54C), with manymutants showing reduction in the number of off-targets (FIG. 61C, 62A).We further explored the surrounding motifs of off-targets forspecificity mutants, and found that REPAIRv1 and most of the engineeredmutants exhibited a strong 3′ G preference for their edits, in agreementwith the characterized ADAR2 motif (FIG. 63A)(26). We selected themutant ADAR2_(DD)(E488Q/T375G) for future experiments, as it had thehighest percent editing of the four mutants with the lowest numbers oftranscriptome-wide off targets and termed it REPAIRv2. Compared toREPAIRv1, REPAIRv2 exhibited increased specificity, with a reductionfrom 1732 to 10 transcriptome off-targets (FIG. 54D). In the regionsurrounding the targeted adenosine in Cluc, REPAIRv2 had reducedoff-target editing, visible in sequencing traces (FIG. 54E). In motifsderived from next-generation sequencing, REPAIRv1 presented a strongpreference towards 3′ G, but showed off-targeting edits for all motifs(FIG. 63B); by contrast, REPAIRv2 only edited the strongest off-targetmotifs (FIG. 63C). The distribution of edits on transcripts was heavilyskewed, with highly-edited genes having over 60 edits (FIG. 64A, 64B),whereas REPAIRv2 only edited one transcript (EEF1A1) multiple times(FIG. 64D-64F). REPAIRv1 off-target edits were predicted to result innumerous variants, including 1000 missense mutations (FIG. 64C) with 93oncogenic events (FIG. 64D). In contrast, REPAIRv2 only had 6 missensemutations (FIG. 64E), none of which had oncogenic consequences (FIG.64F). This reduction in predicted off-target effects distinguishesREPAIRv2 from other RNA editing approaches.

We targeted REPAIRv2 to endogenous genes to test if thespecificity-enhancing mutations reduced nearby edits in targettranscripts while maintaining high-efficiency on-target editing. Forguides targeting either KRAS or PPIB, we found that REPAIRv2 had nodetectable off-target edits, unlike REPAIRv1, and could effectively editthe on-target adenosine at 27.1% and 13%, respectively (FIG. 54F). Thisspecificity extended to additional target sites, including regions thatdemonstrate high-levels of background in non-targeting conditions forREPAIRv1, such as other KRAS or PPIB target sites (FIGS. 65A-65C).Overall, REPAIRv2 eliminated off-targets in duplexed regions around theedited adenosine and showed dramatically enhanced transcriptome-widespecificity.

CONCLUSION

We have shown here that the RNA-guided RNA-targeting type VI-B effectorCas13b is capable of highly efficient and specific RNA knockdown,providing the basis for improved tools for interrogating essential genesand non-coding RNA as well as controlling cellular processes at thetranscriptomic level. Catalytically inactive Cas13b (dCas13b) retainsprogrammable RNA binding capability, which we leveraged here by fusingdCas13b to the adenosine deaminase ADAR2 to achieve precise A to Iedits, a system we term REPAIRv1 (RNA Editing for Programmable A to IReplacement version 1). Further engineering of the system producedREPAIRv2, a method with comparable or increased activity relative tocurrent editing platforms with dramatically improved specificity.

Although Cas13b exhibits high fidelity, our initial results withdCas13b-ADAR2DD fusions revealed thousands of off-targets. To addressthis, we employed a rational mutagenesis strategy to vary the ADAR2_(DD)residues that contact the RNA duplex, identifying a variant,ADAR2_(DD)(E488Q/T375G), capable of precise, efficient, and highlyspecific editing when fused to dCas13b. Editing efficiency with thisvariant was comparable to or better than that achieved with twocurrently available systems, BoxB-ADAR_(DD) or ADAR2 editing. Moreover,the REPAIRv2 system created only 10 observable off-targets in the wholetranscriptome, at least an order of magnitude better than bothalternative editing technologies.

The REPAIR system offers many advantages compared to other nucleic acidediting tools. First, the exact target site can be encoded in the guideby placing a cytidine within the guide extension across from the desiredadenosine to create a favorable A-C mismatch ideal for ADAR editingactivity. Second, Cas13 has no targeting sequence constraints, such as aPFS or PAM, and no motif preference surrounding the target adenosine,allowing any adenosine in the transcriptome to be potentially targetedwith the REPAIR system. We do note, however, that DNA base editors cantarget either the sense or anti-sense strand, while the REPAIR system islimited to transcribed sequences, thereby constraining the total numberof possible editing sites we could target. However, due to the moreflexible nature of targeting with REPAIR, this system can effect moreedits within ClinVar (FIG. 52C) than Cas9-DNA base editors. Third, theREPAIR system directly deaminates target adenosines to inosines and doesnot rely on endogenous repair pathways, such as base-excision ormismatch repair, to generate desired editing outcomes. Thus, REPAIRshould be possible in non-dividing cells that cannot support other formsof editing. Fourth, RNA editing can be transient, allowing the potentialfor temporal control over editing outcomes. This property will likely beuseful for treating diseases caused by temporary changes in cell state,such as local inflammation.

The REPAIR system provides multiple opportunities for additionalengineering. Cas13b possesses pre-crRNA processing activity(13),allowing for multiplex editing of multiple variants, which alone mightnot alter disease risk, but together might have additive effects anddisease-modifying potential. Extension of our rational design approach,such as combining promising mutations, could further increase thespecificity and efficiency of the system, while unbiased screeningapproaches could identify additional residues for improving REPAIRactivity and specificity.

Currently, the base conversions achievable by REPAIR are limited togenerating inosine from adenosine; additional fusions of dCas13 withother catalytic RNA editing domains, such as APOBEC, could enablecytidine to uridine editing. Additionally, mutagenesis of ADAR couldrelax the substrate preference to target cytidine, allowing for theenhanced specificity conferred by the duplexed RNA substrate requirementto be exploited by C→U editors. Adenosine to inosine editing on DNAsubstrates may also be possible with catalytically inactiveDNA-targeting CRISPR effectors, such as dCas9 or dCpf1, either throughformation of DNA-RNA heteroduplex targets(27) or mutagenesis of the ADARdomain.

REPAIR could be applied towards a range of therapeutic indications whereA to I (A to G) editing can reverse or slow disease progression (FIGS.66A-66G). First, expression of REPAIR for targeting causal, Mendelian Gto A mutations in disease-relevant tissues could be used to revertdeleterious mutations and treat disease. For example, stable REPAIRexpression via AAV in brain tissue could be used to correct the GRIN2Amissense mutation c.2191G>A (Asp731Asn) that causes focal epilepsy(28)or the APP missense mutation c.2149G>A (Val717Ile) causing early-onsetAlzheimer's disease(29). Second, REPAIR could be used to treat diseaseby modifying the function of proteins involved in disease-related signaltransduction. For instance, REPAIR editing would allow the re-coding ofsome serine, threonine and tyrosine residues that are the targets ofkinases (FIGS. 66A-66G). Phosphorylation of these residues indisease-relevant proteins affects disease progression for many disordersincluding Alzheimer's disease and multiple neurodegenerativeconditions(30). Third, REPAIR could be used to change the sequence ofexpressed, risk-modifying G to A variants to pre-emptively decrease thechance of entering a disease state for patients. The most intriguingcase are the ‘protective’ risk-modifying alleles, which dramaticallydecrease the chance of entering a disease state, and in some cases,confer additional health benefits. For instance, REPAIR could be used tofunctionally mimic A to G alleles of PCSK9 and IFIH1 that protectagainst cardiovascular disease and psoriatic arthritis(31),respectively. Last, REPAIR can be used to therapeutically modify spliceacceptor and donor sites for exon modulation therapies. REPAIR canchange AU to IU or AA to AI, the functional equivalent of the consensus5′ splice donor or 3′ splice acceptor sites respectively, creating newsplice junctions. Additionally, REPAIR editing can mutate the consensus3′ splice acceptor site from AG->IG to promote skipping of the adjacentdownstream exon, a therapeutic strategy that has received significantinterest for the treatment of DMD. Modulation of splice sites could havebroad applications in diseases where anti-sense oligos have had somesuccess, such as for modulation of SMN2 splicing for treatment of spinalmuscular atrophy(32).

We have demonstrated the use of the PspCas13b enzyme as both an RNAknockdown and RNA editing tool. The dCas13b platform for programmableRNA binding has many applications, including live transcript imaging,splicing modification, targeted localization of transcripts, pull downof RNA-binding proteins, and epitranscriptomic modifications. Here, weused dCas13 to create REPAIR, adding to the existing suite of nucleicacid editing technologies. REPAIR provides a new approach for treatinggenetic disease or mimicking protective alleles, and establishes RNAediting as a useful tool for modifying genetic function. Table 13

TABLE 13 Cas13 Orthologs used in this study Cas13 Cas13 ID abbreviationHost Organism Protein Accession Cas13a1 LshCas13a Leptotrichia shahiiWP_018451595.1 Cas13a2 LwaCas13a Leptotrichia wadei (Lw2) WP_021746774.1Cas13a3 LseCas13a Listeria seeligeri WP_012985477.1 Cas13a4 LbmCas13aLachnospiraceae bacterium WP_044921188.1 MA2020 Cas13a5 LbnCas13aLachnospiraceae bacterium WP_022785443.1 NK4A179 Cas13a6 CamCas13a[Clostridium] aminophilum DSM WP_031473346.1 10710 Cas13a7 CgaCas13aCarnobacterium gallinarum DSM WP_034560163.1 4847 Cas13a8 Cga2Cas13aCarnobacterium gallinarum DSM WP_034563842.1 4847 Cas13a9 Pprcas13aPaludibacter propionicigenes WP_013443710.1 WB4 Cas13a10 LweCas13aListeria weihenstephanensis FSL WP_036059185.1 R9-0317 Cas13a11LbfCas13a Listeriaceae bacterium FSL M6- WP_036091002.1 0635 Cas13a12Lwa2Cas13a Leptotrichia wadei F0279 WP_021746774.1 Cas13a13 RcsCas13aRhodobacter capsulatus SB 1003 WP_013067728.1 Cas13a14 RcrCas13aRhodobacter capsulatus R121 WP_023911507.1 Cas13a15 RcdCas13aRhodobacter capsulatus DE442 WP_023911507.1 Cas13a16 LbuCas13aLeptotrichia buccalis C-1013-b WP_015770004.1 Cas13a17 HheCas13aHerbinix hemicellulosilytica CRZ35554.1 Cas13a18 EreCas13a [Eubacterium]rectale WP_055061018.1 Cas13a19 EbaCas13a Eubacteriaceae bacteriumWP_090127496.1 CHKCI004 Cas13a20 BmaCas13a Blautia sp. Marseille-P2398WP_062808098.1 Cas13a21 LspCas13a Leptotrichia sp. oral taxon 879WP_021744063.1 str. F0557 Cas13b1 BzoCas13b Bergeyella zoohelcumWP_002664492 Cas13b2 PinCas13b Prevotella intermedia WP_036860899Cas13b3 PbuCas13b Prevotella buccae WP_004343973 Cas13b4 AspCas13bAlistipes sp. ZOR0009 WP_047447901 Cas13b5 PsmCas13b Prevotella sp.MA2016 WP_036929175 Cas13b6 RanCas13b Riemerella anatipestiferWP_004919755 Cas13b7 PauCas13b Prevotella aurantiaca WP_025000926Cas13b8 PsaCas13b Prevotella saccharolytica WP_051522484 Cas13b9Pin2Cas13b Prevotella intermedia WP_061868553 Cas13b10 CcaCas13bCapnocytophaga canimorsus WP_013997271 Cas13b11 PguCas13b Porphyromonasgulae WP_039434803 Cas13b12 PspCas13b Prevotella sp. P5-125 WP_044065294Cas13b13 FbrCas13b Flavobacterium branchiophilum WP_014084666 Cas13b14PgiCas13b Porphyromonas gingivalis WP_053444417 Cas13b15 Pin3Cas13bPrevotella intermedia WP_050955369 Cas13c1 FnsCas13c Fusobacteriumnecrophorum WP_005959231.1 subsp. funduliforme ATCC 51357 contig00003Cas13c2 FndCas13c Fusobacterium necrophorum DJ- WP_035906563.1 2contig0065, whole genome shotgun sequence Cas13c3 FnbCas13cFusobacterium necrophorum WP_035935671.1 BFTR-1 contig0068 Cas13c4FnfCas13c Fusobacterium necrophorum EHO19081.1 subsp. funduliforme1_1_36S cont1.14 Cas13c5 FpeCas13c Fusobacterium perfoetens ATCCWP_027128616.1 29250 T364DRAFT_scaffold00009.9_C Cas13c6 FulCas13cFusobacterium ulcerans ATCC WP_040490876.1 49185 cont2.38 Cas13c7AspCas13c Anaerosalibacter sp. ND1 WP_042678931.1 genome assemblyAnaerosalibacter massiliensis ND1

TABLE 14 PFS cutoffs in bacterial screens −Log₂ depletion score used toCas13b ortholog Key generate PFS motif Bergeyella zoohelcum 1 2Prevotella intermedia locus 1 2 1 Prevotella buccae 3 3 Alistipes sp.ZOR0009 4 1 Prevotella sp. MA2016 5 2 Riemerella anatipestifer 6 4Prevotella aurantiaca 7 1 Prevotella saccharolytica 8 0 Prevotellaintermedia locus 2 9 0 Capnocytophaga canimorsus 10 3 Porphyromonasgulae 11 4 Prevotella sp. P5-125 12 2.1 Flavobacterium 13 1branchiophilum Porphyromonas gingivalis 14 3 Prevotella intermedia locus2 15 4

TABLE 15 dCas13b-ADAR linker sequences used in this studyfor RNA editing in mammalian cells. FIG. linker 50CGSGGGGS (SEQ ID NO: 774) 50E GS 57B GSGGGGS 57C GS 57D GS 57E: GS GS57E: GSGGGGS GSGGGGS 57E: (GGGS)3 GGGGSGGGGSGGGGS(SEQ ID NO: 1)57E: Rigid EAAAK(SEQ ID NO: 297) 57E: (GGS)6GGSGGSGGSGGSGGSGGS(SEQ ID NO: 298)) 57E: XTENSGSETPGTSESATPES(SEQ ID NO: 299) 51B GS 57F GS 51C GS 52B GS 52D GS 52EGS 51A: Δ984-1090, Δ1026-1090, Δ1053-1090 GS 51A: Δ1-125, Δ1-88, Δ1-72GSGGGGS (SEQ ID NO: 774) 53B GS 53C GS 53D GS 60A GS 60C GS 60D GS 61DGS 54A GS 62A GS 54B GS 62B GS 62C GS 63A GS 63B GS 54C GS 54D GS 54E GS54F GS 66A GS 66A GS

TABLE 16 Disease information for disease-relevant mutations Gene DiseaseFull length candidates NM_000054.4(AVPR2): c.878G > A AVPR2 Nephrogenicdiabetes insipidus, (p.Trp293Ter) X-linked NM_000136.2(FANCC): c.1517G >A FANCC Fanconi anemia, (p.Trp506Ter) complementation group C Additionalsimulated candiates Candidate NM_000206.2(IL2RG): c.710G > A IL2RGX-linked severe combined (p.Trp237Ter) immunodeficiency NM_000132.3(F8):c.3144G > A F8 Hereditary factor VIII (p.Trp1048Ter) deficiency diseaseNM_000527.4(LDLR): c.1449G > A LDLR Familial hypercholesterolemia(p.Trp483Ter) NM_000071.2(CBS): c.162G > A CBS Homocystinuria due to CBS(p.Trp54Ter) deficiency NM_000518.4(HBB): c.114G > A HBB beta{circumflexover ( )}0{circumflex over ( )} Thalassemia|beta (p.Trp38Ter)Thalassemia NM_000035.3(ALDOB): c.888G > A ALDOB Hereditary fructosuria(p.Trp296Ter) NM_004006.2(DMD): c.3747G > A DMD Duchenne musculardystrophy (p.Trp1249Ter) NM_005359.5(SMAD4): c.906G > A SMAD4 Juvenilepolyposis syndrome (p.Trp302Ter) NM_000059.3(BRCA2): c.582G > A BRCA2Familial cancer of breast|Breast- (p.Trp194Ter) ovarian cancer, familial2 NM_000833.4(GRIN2A): c.3813G > A GRIN2A Epilepsy, focal, with speech(p.Trp1271Ter) disorder and with or without mental retardationNM_002977.3(SCN9A): c.2691G > A SCN9A Indifference to pain, congenital,(p.Trp897Ter) autosomal recessive NM_007375.3(TARDBP): c.943G > A TARDBPAmyotrophic lateral sclerosis (p.Ala315Thr) type 10 NM_000492.3(CFTR):c.3846G > A CFTR Cystic fibrosis|Hereditary (p.Trp1282Ter)pancreatitis|not provided|ataluren response - EfficacyNM_130838.1(UBE3A): c.2304G > A UBE3A Angelman syndrome (p.Trp768Ter)NM_000543.4(SMPD1): c.168G > A SMPD1 Niemann-Pick disease, type A(p.Trp56Ter) NM_206933.2(USH2A): c.9390G > A USH2A Usher syndrome, type2A (p.Trp3130Ter) NM_130799.2(MEN1): c.1269G > A MEN1 Hereditarycancer-predisposing (p.Trp423Ter) syndrome NM_177965.3(C8orf37):c.555G > A C8orf37 Retinitis pigmentosa 64 (p.Trp185Ter)NM_000249.3(MLH1): c.1998G > A MLH1 Lynch syndrome (p.Trp666Ter)NM_000548.4(TSC2): c.2108G > A TSC2 Tuberous sclerosis 2|Tuberous(p.Trp703Ter) sclerosis syndrome NM_000267.3(NF1): c.7044G > A NF1Neurofibromatosis, type 1 (p.Trp2348Ter) NM_000179.2(MSH6): c.3020G > AMSH6 Lynch syndrome (p.Trp1007Ter) NM_000344.3(SMN1): c.305G > A SMN1Spinal muscular atrophy, type (p.Trp102Ter) II|Kugelberg-Welanderdisease NM_024577.3(SH3TC2): c.920G > A SH3TC2 Charcot-Marie-Toothdisease, (p.Trp307Ter) type 4C NM_001369.2(DNAH5): c.8465G > A DNAH5Primary ciliary dyskinesia (p.Trp2822Ter) NM_004992.3(MECP2): c.311 G >A MECP2 Rett syndrome (p.Trp104Ter) NM_032119.3(ADGRV1): c.7406G > AADGRV1 Usher syndrome, type 2C (p.Trp2469Ter) NM_017651.4(AHI1):c.2174G > A AHI1 Joubert syndrome 3 (p.Trp725Ter) NM_004562.2(PRKN):c.1358G > A PRKN Parkinson disease 2 (p.Trp453Ter) NM_000090.3(COL3A1):c.3833G > A COL3A1 Ehlers-Danlos syndrome, type 4 (p.Trp1278Ter)NM_007294.3(BRCA1): c.5511G > A BRCA1 Familial cancer of breast|Breast-(p.Trp1837Ter) ovarian cancer, familial 1 NM_000256.3(MYBPC3): c.3293G >A MYBPC3 Primary familial hypertrophic (p.Trp1098Ter) cardiomyopathyNM_000038.5(APC): c.1262G > A APC Familial adenomatous polyposis(p.Trp421Ter) 1 NM_001204.6(BMPR2): c.893G > A BMPR2 Primary pulmonary(p.W298*) hypertension

TABLE 17 Key plasmids used in this study Plasmid name DescriptionpAB0006 CMV-Cluciferase-polyA EF1a-G-luciferase-poly A pAB0040CMV-Cluciferase(STOP85)-polyA EF1a-G-luciferase- polyA pAB0048pCDNA-ADAR2-N-terminal B12-HIV NES pAB0050 pAB0001-A02 (crRNA backbone)pAB0051 pAB0001-B06 (crRNA backbone) pAB0052 pAB0001-B11 (crRNAbackbone) pAB0053 pAB0001-B12 (crRNA backbone) pAB0014.B6EF1a-BsiWI-Cas13b6-NES-mapk pAB0013.B11 EF1a-BsiWI-Cas13b11-NES-HIVpAB0013.B12 EF1a-BsiWI-Cas13b12-NES-HIV pAB0051 pAB0001-B06 (crRNAbackbone) pAB0052 pAB0001-B11 (crRNA backbone) pAB0053 pAB0001-B12(crRNA backbone) pAB0079 pCDNA-ADAR1hu-EQmutant-N-terminal destinationvector pAB0085 pCDNA-ADAR2 (E488Q)hu-EQmutant-N-terminal destinationvector pAB0095 EF1a-BsiWI-Cas13-B12-NES-HIV, with double H HEPN mutantpAB0114 pCDNA-wtADAR2hu-EQmutant-N-terminal destination vector pAB0120Luciferase ADAR guide optimal (guide 24 from wC0054) pAB0122 pAB0001-B12NT guide for ADAR experiments pAB0151 pCDNA-ADAR2hu-EQmutant-N-terminaldestination vector C-term delta 984-1090 pAB0180 T375G specificitymutant pAB0181 T375G Cas13b C-term delta 984-1090

TABLE 18 Guide/shRNA sequences used in this studyfor knockdown in mammalian cells Interference Name Spacer sequenceMechanism Notes First FIG. Bacterial GCCAGCUUUCCGGGCAUUGGCUUCC Cas13bUsed for all PFS guide AUC orthologs (SEQ ID NO: 300) Cas13a-GCCAGCTTTCCGGGCATTGGCTTCC Cas13a Used for all FIG. 49B Gluc ATCCas13a orthologs guide 1 (SEQ ID NO: 301) Cas13a-ACCCAGGAATCTCAGGAATGTCGAC Cas13a Used for all FIG. 49B Gluc GATCas13a orthologs guide 2 (SEQ ID NO: 302) Cas13a-AGGGTTTTCCCAGTCACGACGTTGT Cas13a Used for all FIG. 49B non- AAACas13a orthologs targeting (SEQ ID NO: 303) guide (LacZ) Cas13b-GGGCATTGGCTTCCATCTCTTTGAG Cas13b Used for orthologs FIG. 49B Gluc CACCT1-3, 6, 7, 10, 11, guide 1.1 (SEQ ID NO: 304) 12, 14, 15 Cas13b-GUGCAGCCAGCUUUCCGGGCAUUGG Cas13b Used for ortholog FIG. 49B Gluc CUUCC 4guide 1.2 (SEQ ID NO: 305) Cas13b- GCAGCCAGCUUUCCGGGCAUUGGCU Cas13bUsed for ortholog FIG. 49B Gluc UCCAU 5 guide 1.3 (SEQ ID NO: 306)Cas13b- GGCUUCCAUCUCUUUGAGCACCUCC Cas13b Used for ortholog FIG. 49B GlucAGCGG 8, 9 guide 1.4 (SEQ ID NO: 307) Cas13b- GGAAUGUCGACGAUCGCCUCGCCUACas13b Used for ortholog FIG. 49B Gluc UGCCG 13 guide 1.5(SEQ ID NO: 308) Cas13b- GAAUGUCGACGAUCGCCUCGCCUAU Cas13bUsed for orthologs FIG. 49B Gluc GCCGC 1-3, 6, 7, 10, 11, guide 2.1(SEQ ID NO: 309) 14, 15 Cas13b- GACCUGUGCGAUGAACUGCUCCAUG Cas13bUsed for ortholog FIG. 49B Gluc GGCUC 12 guide 2.2 (SEQ ID NO: 310)Cas13b- GUGUGGCAGCGUCCUGGGAUGAACU Cas13b Used for ortholog FIG. 49B GlucUCUUC 4 guide 2.2 (SEQ ID NO: 311) Cas13b- GUGGCAGCGUCCUGGGAUGAACUUCCas13b Used for ortholog FIG. 49B Gluc UUCAU 5 guide 2.3(SEQ ID NO: 312) Cas13b- GCUUCUUGCCGGGCAACUUCCCGCG Cas13bUsed for ortholog FIG. 49B Gluc GUCAG 8, 9 guide 2.4 (SEQ ID NO: 313)Cas13b- GCAGGGUUUUCCCAGUCACGACGUU Cas13b Used for ortholog FIG. 49B GlucGUAAAA 13 guide 2.6 (SEQ ID NO: 314) Cas13b- GCAGGGUUUUCCCAGUCACGACGUUCas13b Used for all FIG. 49B non GUAAAA orthologs targeting(SEQ ID NO: 315) guide Cas13a- ACCCAGGAAUCUCAGGAAUGUCGAC Cas13a FIG. 49EGluc GAU guide- (SEQ ID NO: 316) RNASeq shRNA- CAGCTTTCCGGGCATTGGCTTshRNA FIG. 49F Gluc (SEQ ID NO: 317) guide Cas13b-CCGCUGGAGGUGCUCAAAGAGAUGG Cas13b FIG. 49F Gluc AAGCC guide-(SEQ ID NO: 318) RNASeq Cas13a- GCCAGCTTTCCGGGCATTGGCTTCC Cas13aFIG. 56A Gluc- ATC guide-1 (SEQ ID NO: 319) Cas13a-ACCCAGGAATCTCAGGAATGTCGAC Cas13a FIG. 56A Gluc- GAT guide-2(SEQ ID NO: 320) Cas13b- GGGCATTGGCTTCCATCTCTTTGAG Cas13b FIG. 56AGluc-opt- CACCT guide-1 (SEQ ID NO: 321) Cas13b-GAAUGUCGACGAUCGCCUCGCCUAU Cas13b FIG. 56A Gluc-opt- GCCGC guide-2(SEQ ID NO: 322) Cas13a CAAGGCACTCTTGCCTACGCCACCA Cas13a FIG. 56B KRASGCT guide 1 (SEQ ID NO: 323) Cas13a TCATATTCGTCCACAAAATGATTCT Cas13aFIG. 56B KRAS GAA guide 2 (SEQ ID NO: 324) Cas13aATTATTTATGGCAAATACACAAAGA Cas13a FIG. 56B KRAS AAG guide 3(SEQ ID NO: 325) Cas13a GAATATCTTCAAATGATTTAGTATT Cas13a FIG. 56B KRASATT guide 4 (SEQ ID NO: 326) Cas13a ACCATAGGTACATCTTCAGAGTCCT Cas13aFIG. 56B KRAS TAA guide 5 (SEQ ID NO: 327) Cas13bGTCAAGGCACTCTTGCCTACGCCAC Cas13b FIG. 56B KRAS CAGCT guide 1(SEQ ID NO: 328) Cas13b GATCATATTCGTCCACAAAATGATT Cas13b FIG. 56B KRASCTGAA guide 2 (SEQ ID NO: 329) Cas13b GTATTATTTATGGCAAATACACAAA Cas13bFIG. 56B KRAS GAAAG guide 3 (SEQ ID NO: 330) Cas13bGTGAATATCTTCAAATGATTTAGTA Cas13b FIG. 56B KRAS TTATT guide 4(SEQ ID NO: 331) Cas13b GGACCATAGGTACATCTTCAGAGTC Cas13b FIG. 56B KRASCTTAA guide 5 (SEQ ID NO: 332) shRNA aagagtgccttgacgatacagcCTC shRNAFIG. 56B KRAS GAGgctgtatcgtcaaggcactctt guide 1 (SEQ ID NO: 333) shRNAaatcattttgtggacgaatatCTCG shRNA FIG. 56B KRAS AGatattcgtccacaaaatgattguide 2 (SEQ ID NO: 334) shRNA aaataatactaaatcatttgaCTCG shRNA FIG. 56BKRAS AGtcaaatgatttagtattattt guide 3 (SEQ ID NO: 335) shRNAaataatactaaatcatttgaaCTCG shRNA FIG. 56B KRAS AGttcaaatgatttagtattattguide 4 (SEQ ID NO: 336) shRNA aaggactctgaagatgtacctCTCG shRNA FIG. 56BKRAS AGaggtacatcttcagagtcctt guide 5 (SEQ ID NO: 337)

TABLE 19 Guide sequences used for Gluc knockdown First NameSpacer sequence Position Notes FIG. Gluc tilingGAGATCAGGGCAAACAGAACTTTGACTCCC   2 Note that the Cas13a spacers are 49Cguide 1 (SEQ ID NO: 338) are truncated by two nucleotides at the 5′ endGluc tiling GGATGCAGATCAGGGCAAACAGAACTTTGA   7Note that the Cas13a spacers 49C guide 2 (SEQ ID NO: 339)are truncated by two nucleotides at the 5′ end Gluc tilingGCACAGCGATGCAGATCAGGGCAAACAGAA  13 Note that the Cas13a spacers 49Cguide 3 (SEQ ID NO: 340) are truncated by two nucleotides at the 5′ endGluc tiling GCTCGGCCACAGCGATGCAGATCAGGGCAA  19Note that the Cas13a spacers 49C guide 4 (SEQ ID NO: 341)are truncated by two nucleotides at the 5′ end Gluc tilingGGGGCTTGGCCTCGGCCACAGCGATGCAGA  28 Note that the Cas13a spacers 49Cguide 5 (SEQ ID NO: 342) are truncated by two nucleotides at the 5′ endGluc tiling GTGGGCTTGGCCTCGGCCACAGCGATGCAG  29Note that the Cas13a spacers 49C guide 6 (SEQ ID NO: 343)are truncated by two nucleotides at the 5′ end Gluc tilingGTCTCGGTGGGCTTGGCCTCGGCCACAGCG  35 Note that the Cas13a spacers 49Cguide 7 (SEQ ID NO: 344) are truncated by two nucleotides at the 5′ endGluc tiling GTTCGTTGTTCTCGGTGGGCTTGGCCTCGG  43Note that the Cas13a spacers 49C guide 8 (SEQ ID NO: 345)are truncated by two nucleotides at the 5′ end Gluc tilingGGAAGTCTTCGTTGTTCTCGGTGGGCTTGG  49 Note that the Cas13a spacers 49Cguide 9 (SEQ ID NO: 346) are truncated by two nucleotides at the 5′ endGluc tiling GATGTTGAAGTCTTCGTTGTTCTCGGTGGG  54Note that the Cas13a spacers 49C guide 10 (SEQ ID NO: 347)are truncated by two nucleotides at the 5′ end Gluc tilingGCGGCCACGATGTTGAAGTCTTCGTTGTTC  62 Note that the Cas13a spacers 49Cguide 11 (SEQ ID NO: 348) are truncated by two nucleotides at the 5′ endGluc tiling GTGGCCACGGCCACGATGTTGAAGTCTTCG  68Note that the Cas13a spacers 49C guide 12 (SEQ ID NO: 349)are truncated by two nucleotides at the 5′ end Gluc tilingGGTTGCTGGCCACGGCCACGATGTTGAAGT  73 Note that the Cas13a spacers 49Cguide 13 (SEQ ID NO: 350) are truncated by two nucleotides at the 5′ endGluc tiling GTCGCGAAGTTGCTGGCCACGGCCACGATG  80Note that the Cas13a spacers 49C guide 14 (SEQ ID NO: 351)are truncated by two nucleotides at the 5′ end Gluc tilingGCCGTGGTCGCGAAGTTGCTGGCCACGGCC  86 Note that the Cas13a spacers 49Cguide 15 (SEQ ID NO: 352) are truncated by two nucleotides at the 5′ endGluc tiling GCGAGATCCGTGGTCGCGAAGTTGCTGGCC  92Note that the Cas13a spacers 49C guide 16 (SEQ ID NO: 353)are truncated by two nucleotides at the 5′ end Gluc tilingGCAGCATCGAGATCCGTGGTCGCGAAGTTG  98 Note that the Cas13a spacers 49Cguide 17 (SEQ ID NO: 354) are truncated by two nucleotides at the 5′ endGluc tiling GGGTCAGCATCGAGATCCGTGGTCGCGAAG 101Note that the Cas13a spacers 49C guide 18 (SEQ ID NO: 355)are truncated by two nucleotides at the 5′ end Gluc tilingGCTTCCCGCGGTCAGCATCGAGATCCGTGG 109 Note that the Cas13a spacers 49Cguide 19 (SEQ ID NO: 356) are truncated by two nucleotides at the 5′ endGluc tiling GGGGCAACTTCCCGCGGTCAGCATCGAGAT 115Note that the Cas13a spacers 49C guide 20 (SEQ ID NO: 357)are truncated by two nucleotides at the 5′ end Gluc tilingGTCTTGCCGGGCAACTTCCCGCGGTCAGCA 122 Note that the Cas13a spacers 49Cguide 21 (SEQ ID NO: 358) are truncated by two nucleotides at the 5′ endGluc tiling GGCAGCTTCTTGCCGGGCAACTTCCCGCGG 128Note that the Cas13a spacers 49C guide 22 (SEQ ID NO: 359)are truncated by two nucleotides at the 5′ end Gluc tilingGCCAGCGGCAGCTTCTTGCCGGGCAACTTC 134 Note that the Cas13a spacers 49Cguide 23 (SEQ ID NO: 360) are truncated by two nucleotides at the 5′ endGluc tiling GCACCTCCAGCGGCAGCTTCTTGCCGGGCA 139Note that the Cas13a spacers 49C guide 24 (SEQ ID NO: 361)are truncated by two nucleotides at the 5′ end Gluc tilingGCTTTGAGCACCTCCAGCGGCAGCTTCTTG 146 Note that the Cas13a spacers 49Cguide 25 (SEQ ID NO: 362) are truncated by two nucleotides at the 5′ endGluc tiling GCATCTCTTTGAGCACCTCCAGCGGCAGCT 151Note that the Cas13a spacers 49C guide 26 (SEQ ID NO: 363)are truncated by two nucleotides at the 5′ end Gluc tilingGTCCATCTCTTTGAGCACCTCCAGCGGCAG 153 Note that the Cas13a spacers 49Cguide 27 (SEQ ID NO: 364) are truncated by two nucleotides at the 5′ endGluc tiling GGGCATTGGCTTCCATCTCTTTGAGCACCT 163Note that the Cas13a spacers 49C guide 28 (SEQ ID NO: 365)are truncated by two nucleotides at the 5′ end Gluc tilingGTCCGGGCATTGGCTTCCATCTCTTTGAGC 167 Note that the Cas13a spacers 49Cguide 29 (SEQ ID NO: 366) are truncated by two nucleotides at the 5′ endGluc tiling GGCCAGCTTTCCGGGCATTGGCTTCCATCT 175Note that the Cas13a spacers 49C guide 30 (SEQ ID NO: 367)are truncated by two nucleotides at the 5′ end Gluc tilingGGGTGCAGCCAGCTTTCCGGGCATTGGCTT 181 Note that the Cas13a spacers 49Cguide 31 (SEQ ID NO: 368) are truncated by two nucleotides at the 5′ endGluc tiling GAGCCCCTGGTGCAGCCAGCTTTCCGGGCA 188Note that the Cas13a spacers 49C guide 32 (SEQ ID NO: 369)are truncated by two nucleotides at the 5′ end Gluc tilingGATCAGACAGCCCCTGGTGCAGCCAGCTTT 195 Note that the Cas13a spacers 49Cguide 33 (SEQ ID NO: 370) are truncated by two nucleotides at the 5′ endGluc tiling GGCAGATCAGACAGCCCCTGGTGCAGCCAG 199Note that the Cas13a spacers 49C guide 34 (SEQ ID NO: 371)are truncated by two nucleotides at the 5′ end Gluc tilingGACAGGCAGATCAGACAGCCCCTGGTGCAG 203 Note that the Cas13a spacers 49Cguide 35 (SEQ ID NO: 372) are truncated by two nucleotides at the 5′ endGluc tiling GTGATGTGGGACAGGCAGATCAGACAGCCC 212Note that the Cas13a spacers 49C guide 36 (SEQ ID NO: 373)are truncated by two nucleotides at the 5′ end Gluc tilingGACTTGATGTGGGACAGGCAGATCAGACAG 215 Note that the Cas13a spacers 49Cguide 37 (SEQ ID NO: 374) are truncated by two nucleotides at the 5′ endGluc tiling GGGGCGTGCACTTGATGTGGGACAGGCAGA 223Note that the Cas13a spacers 49C guide 38 (SEQ ID NO: 375)are truncated by two nucleotides at the 5′ end Gluc tilingGCTTCATCTTGGGCGTGCACTTGATGTGGG 232 Note that the Cas13a spacers 49Cguide 39 (SEQ ID NO: 376) are truncated by two nucleotides at the 5′ endGluc tiling GTGAACTTCTTCATCTTGGGCGTGCACTTG 239Note that the Cas13a spacers 49C guide 40 (SEQ ID NO: 377)are truncated by two nucleotides at the 5′ end Gluc tilingGGGATGAACTTCTTCATCTTGGGCGTGCAC 242 Note that the Cas13a spacers 49Cguide 41 (SEQ ID NO: 378) are truncated by two nucleotides at the 5′ endGluc tiling GTGGGATGAACTTCTTCATCTTGGGCGTGC 244Note that the Cas13a spacers 49C guide 42 (SEQ ID NO: 379)are truncated by two nucleotides at the 5′ end Gluc tilingGGGCAGCGTCCTGGGATGAACTTCTTCATC 254 Note that the Cas13a spacers 49Cguide 43 (SEQ ID NO: 380) are truncated by two nucleotides at the 5′ endGluc tiling GGGTGTGGCAGCGTCCTGGGATGAACTTCT 259Note that the Cas13a spacers 49C guide 44 (SEQ ID NO: 381)are truncated by two nucleotides at the 5′ end Gluc tilingGTTCGTAGGTGTGGCAGCGTCCTGGGATGA 265 Note that the Cas13a spacers 49Cguide 45 (SEQ ID NO: 382) are truncated by two nucleotides at the 5′ endGluc tiling GCGCCTTCGTAGGTGTGGCAGCGTCCTGGG 269Note that the Cas13a spacers 49C guide 46 (SEQ ID NO: 383)are truncated by two nucleotides at the 5′ end Gluc tilingGTCTTTGTCGCCTTCGTAGGTGTGGCAGCG 276 Note that the Cas13a spacers 49Cguide 47 (SEQ ID NO: 384) are truncated by two nucleotides at the 5′ endGluc tiling GCTTTGTCGCCTTCGTAGGTGTGGCAGCGT 275Note that the Cas13a spacers 49C guide 48 (SEQ ID NO: 385)are truncated by two nucleotides at the 5′ end Gluc tilingGTGCCGCCCTGTGCGGACTCTTTGTCGCCT 293 Note that the Cas13a spacers 49Cguide 49 (SEQ ID NO: 386) are truncated by two nucleotides at the 5′ endGluc tiling GTATGCCGCCCTGTGCGGACTCTTTGTCGC 295Note that the Cas13a spacers 49C guide 50 (SEQ ID NO: 387)are truncated by two nucleotides at the 5′ end Gluc tilingGCCTCGCCTATGCCGCCCTGTGCGGACTCT 302 Note that the Cas13a spacers 49Cguide 51 (SEQ ID NO: 388) are truncated by two nucleotides at the 5′ endGluc tiling GGATCGCCTCGCCTATGCCGCCCTGTGCGG 307Note that the Cas13a spacers 49C guide 52 (SEQ ID NO: 389)are truncated by two nucleotides at the 5′ end Gluc tilingGATGTCGACGATCGCCTCGCCTATGCCGCC 315 Note that the Cas13a spacers 49Cguide 53 (SEQ ID NO: 390) are truncated by two nucleotides at the 5′ endGluc tiling GCAGGAATGTCGACGATCGCCTCGCCTATG 320Note that the Cas13a spacers 49C guide 54 (SEQ ID NO: 391)are truncated by two nucleotides at the 5′ end Gluc tilingGAATCTCAGGAATGTCGACGATCGCCTCGC 325 Note that the Cas13a spacers 49Cguide 55 (SEQ ID NO: 392) are truncated by two nucleotides at the 5′ endGluc tiling GCCCAGGAATCTCAGGAATGTCGACGATCG 331Note that the Cas13a spacers 49C guide 56 (SEQ ID NO: 393)are truncated by two nucleotides at the 5′ end Gluc tilingGCCTTGAACCCAGGAATCTCAGGAATGTCG 338 Note that the Cas13a spacers 49Cguide 57 (SEQ ID NO: 394) are truncated by two nucleotides at the 5′ endGluc tiling GCCAAGTCCTTGAACCCAGGAATCTCAGGA 344Note that the Cas13a spacers 49C guide 58 (SEQ ID NO: 395)are truncated by two nucleotides at the 5′ end Gluc tilingGTGGGCTCCAAGTCCTTGAACCCAGGAATC 350 Note that the Cas13a spacers 49Cguide 59 (SEQ ID NO: 396) are truncated by two nucleotides at the 5′ endGluc tiling GCCATGGGCTCCAAGTCCTTGAACCCAGGA 353Note that the Cas13a spacers 49C guide 60 (SEQ ID NO: 397)are truncated by two nucleotides at the 5′ end Gluc tilingGGAACTGCTCCATGGGCTCCAAGTCCTTGA 361 Note that the Cas13a spacers 49Cguide 61 (SEQ ID NO: 398) are truncated by two nucleotides at the 5′ endGluc tiling GTGCGATGAACTGCTCCATGGGCTCCAAGT 367Note that the Cas13a spacers 49C guide 62 (SEQ ID NO: 399)are truncated by two nucleotides at the 5′ end Gluc tilingGGACCTGTGCGATGAACTGCTCCATGGGCT 373 Note that the Cas13a spacers 49Cguide 63 (SEQ ID NO: 400) are truncated by two nucleotides at the 5′ endGluc tiling GACAGATCGACCTGTGCGATGAACTGCTCC 380Note that the Cas13a spacers 49C guide 64 (SEQ ID NO: 401)are truncated by two nucleotides at the 5′ end Gluc tilingGACACACAGATCGACCTGTGCGATGAACTG 384 Note that the Cas13a spacers 49Cguide 65 (SEQ ID NO: 402) are truncated by two nucleotides at the 5′ endGluc tiling GTGCAGTCCACACACAGATCGACCTGTGCG 392Note that the Cas13a spacers 49C guide 66 (SEQ ID NO: 403)are truncated by two nucleotides at the 5′ end Gluc tilingGCCAGTTGTGCAGTCCACACACAGATCGAC 399 Note that the Cas13a spacers 49Cguide 67 (SEQ ID NO: 404) are truncated by two nucleotides at the 5′ endGluc tiling GGGCAGCCAGTTGTGCAGTCCACACACAGA 404Note that the Cas13a spacers 49C guide 68 (SEQ ID NO: 405)are truncated by two nucleotides at the 5′ end Gluc tilingGTTTGAGGCAGCCAGTTGTGCAGTCCACAC 409 Note that the Cas13a spacers 49Cguide 69 (SEQ ID NO: 406) are truncated by two nucleotides at the 5′ endGluc tiling GAAGCCCTTTGAGGCAGCCAGTTGTGCAGT 415Note that the Cas13a spacers 49C guide 70 (SEQ ID NO: 407)are truncated by two nucleotides at the 5′ end Gluc tilingGCACGTTGGCAAGCCCTTTGAGGCAGCCAG 424 Note that the Cas13a spacers 49Cguide 71 (SEQ ID NO: 408) are truncated by two nucleotides at the 5′ endGluc tiling GACTGCACGTTGGCAAGCCCTTTGAGGCAG 428Note that the Cas13a spacers 49C guide 72 (SEQ ID NO: 409)are truncated by two nucleotides at the 5′ end Gluc tilingGGGTCAGAACACTGCACGTTGGCAAGCCCT 437 Note that the Cas13a spacers 49Cguide 73 (SEQ ID NO: 410) are truncated by two nucleotides at the 5′ endGluc tiling GCAGGTCAGAACACTGCACGTTGGCAAGCC 439Note that the Cas13a spacers 49C guide 74 (SEQ ID NO: 411)are truncated by two nucleotides at the 5′ end Gluc tilingGAGCAGGTCAGAACACTGCACGTTGGCAAG 441 Note that the Cas13a spacers 49Cguide 75 (SEQ ID NO: 412) are truncated by two nucleotides at the 5′ endGluc tiling GGCCACTTCTTGAGCAGGTCAGAACACTGC 452Note that the Cas13a spacers 49C guide 76 (SEQ ID NO: 413)are truncated by two nucleotides at the 5′ end Gluc tilingGCGGCAGCCACTTCTTGAGCAGGTCAGAAC 457 Note that the Cas13a spacers 49Cguide 77 (SEQ ID NO: 414) are truncated by two nucleotides at the 5′ endGluc tiling GTGCGGCAGCCACTTCTTGAGCAGGTCAGA 459Note that the Cas13a spacers 49C guide 78 (SEQ ID NO: 415)are truncated by two nucleotides at the 5′ end Gluc tilingGAGCGTTGCGGCAGCCACTTCTTGAGCAGG 464 Note that the Cas13a spacers 49Cguide 79 (SEQ ID NO: 416) are truncated by two nucleotides at the 5′ endGluc tiling GAAAGGTCGCACAGCGTTGCGGCAGCCACT 475Note that the Cas13a spacers 49C guide 80 (SEQ ID NO: 417)are truncated by two nucleotides at the 5′ end Gluc tilingGCTGGCAAAGGTCGCACAGCGTTGCGGCAG 480 Note that the Cas13a spacers 49Cguide 81 (SEQ ID NO: 418) are truncated by two nucleotides at the 5′ endGluc tiling GGGCAAAGGTCGCACAGCGTTGCGGCAGCC 478Note that the Cas13a spacers 49C guide 82 (SEQ ID NO: 419)are truncated by two nucleotides at the 5′ end Gluc tilingGTGGATCTTGCTGGCAAAGGTCGCACAGCG 489 Note that the Cas13a spacers 49Cguide 83 (SEQ ID NO: 420) are truncated by two nucleotides at the 5′ endGluc tiling GCACCTGGCCCTGGATCTTGCTGGCAAAGG 499Note that the Cas13a spacers 49C guide 84 (SEQ ID NO: 421)are truncated by two nucleotides at the 5′ end Gluc tilingGTGGCCCTGGATCTTGCTGGCAAAGGTCGC 495 Note that the Cas13a spacers 49Cguide 85 (SEQ ID NO: 422) are truncated by two nucleotides at the 5′ endGluc tiling GTGATCTTGTCCACCTGGCCCTGGATCTTG 509Note that the Cas13a spacers 49C guide 86 (SEQ ID NO: 423)are truncated by two nucleotides at the 5′ end Gluc tilingGCCCCTTGATCTTGTCCACCTGGCCCTGGA 514 Note that the Cas13a spacers 49Cguide 87 (SEQ ID NO: 424) are truncated by two nucleotides at the 5′ endGluc tiling GCCCTTGATCTTGTCCACCTGGCCCTGGAT 513Note that the Cas13a spacers 49C guide 88 (SEQ ID NO: 425)are truncated by two nucleotides at the 5′ end Gluc tilingGCCTTGATCTTGTCCACCTGGCCCTGGATC 512 Note that the Cas13a spacers 49Cguide 89 (SEQ ID NO: 426) are truncated by two nucleotides at the 5′ endGluc tiling GGCAAAGGTCGCACAGCGTTGCGGCAGCCA 477Note that the Cas13a spacers 49C guide 90 (SEQ ID NO: 427)are truncated by two nucleotides at the 5′ end Gluc tilingGCAAAGGTCGCACAGCGTTGCGGCAGCCAC 476 Note that the Cas13a spacers 49Cguide 91 (SEQ ID NO: 428) are truncated by two nucleotides at the 5′ endGluc tiling GAAGGTCGCACAGCGTTGCGGCAGCCACTT 474Note that the Cas13a spacers 49C guide 92 (SEQ ID NO: 429)are truncated by two nucleotides at the 5′ end Gluc tilingGAGGTCGCACAGCGTTGCGGCAGCCACTTC 473 Note that the Cas13a spacers 49Cguide 93 (SEQ ID NO: 430) are truncated by two nucleotides at the 5′ endNon-targeting GGTAATGCCTGGCTTGTCGACGCATAGTCTG N/ANote that the Cas13a spacers 49C guide 1 (SEQ ID NO: 431)are truncated by two nucleotides at the 5′ end Non-targetingGGGAACCTTGGCCGTTATAAAGTCTGACCAG N/A Note that the Cas13a spacers 49Cguide 2 (SEQ ID NO: 432) are truncated by two nucleotides at the 5′ endNon-targeting GGAGGGTGAGAATTTAGAACCAAGATTGTTG N/ANote that the Cas13a spacers 49C guide 3 (SEQ ID NO: 433)are truncated by two nucleotides at the 5′ end

TABLE 20 Guide sequences used for Cluc knockdown First NameSpacer sequence Position Notes FIG. Cluc tilingGAGTCCTGGCAATGAACAGTGGCGCAGTAG   32 Note that the Cas13a spacers 49Dguide 1 (SEQ ID NO: 434) are truncated by two nucleotides at the 5′ endCluc tiling GGGTGCCACAGCTGCTATCAATACATTCTC  118Note that the Cas13a spacers 49D guide 2 (SEQ ID NO: 435)are truncated by two nucleotides at the 5′ end Cluc tilingGTTACATACTGACACATTCGGCAACATGTT  197 Note that the Cas13a spacers 49Dguide 3 (SEQ ID NO: 436) are truncated by two nucleotides at the 5′ endCluc tiling GTATGTACCAGGTTCCTGGAACTGGAATCT  276Note that the Cas13a spacers 49D guide 4 (SEQ ID NO: 437)are truncated by two nucleotides at the 5′ end Cluc tilingGCCTTGGTTCCATCCAGGTTCTCCAGGGTG  350 Note that the Cas13a spacers 49Dguide 5 (SEQ ID NO: 438) are truncated by two nucleotides at the 5′ endCluc tiling GCAGTGATGGGATTCTCAGTAGCTTGAGCG  431Note that the Cas13a spacers 49D guide 6 (SEQ ID NO: 439)are truncated by two nucleotides at the 5′ end Cluc tilingGAGCCTGGCATCTCAACAACAGCGATGGTG  512 Note that the Cas13a spacers 49Dguide 7 (SEQ ID NO: 440) are truncated by two nucleotides at the 5′ endCluc tiling GTGTCTGGGGCGATTCTTACAGATCTTCCT  593Note that the Cas13a spacers 49D guide 8 (SEQ ID NO: 441)are truncated by two nucleotides at the 5′ end Cluc tilingGCTGGATCTGAAGTGAAGTCTGTATCTTCC  671 Note that the Cas13a spacers 49Dguide 9 (SEQ ID NO: 442) are truncated by two nucleotides at the 5′ endCluc tiling GGCAACGTCATCAGGATTTCCATAGAGTGG  747Note that the Cas13a spacers 49D guide 10 (SEQ ID NO: 443)are truncated by two nucleotides at the 5′ end Cluc tilingGAGGCGCAGGAGATGGTGTAGTAGTAGAAG  830 Note that the Cas13a spacers 49Dguide 11 (SEQ ID NO: 444) are truncated by two nucleotides at the 5′ endCluc tiling GAGGGACCCTGGAATTGGTATCTTGCTTTG  986Note that the Cas13a spacers 49D guide 13 (SEQ ID NO: 445)are truncated by two nucleotides at the 5′ end Cluc tilingGGTAAGAGTCAACATTCCTGTGTGAAACCT 1066 Note that the Cas13a spacers 49Dguide 14 (SEQ ID NO: 446) are truncated by two nucleotides at the 5′ endCluc tiling GACCAGAATCTGTTTTCCATCAACAATGAG 1143Note that the Cas13a spacers 49D guide 15 (SEQ ID NO: 447)are truncated by two nucleotides at the 5′ end Cluc tilingGATGGCTGTAGTCAGTATGTCACCATCTTG 1227 Note that the Cas13a spacers 49Dguide 16 (SEQ ID NO: 448) are truncated by two nucleotides at the 5′ endCluc tiling GTACCATCGAATGGATCTCTAATATGTACG 1304Note that the Cas13a spacers 49D guide 17 (SEQ ID NO: 449)are truncated by two nucleotides at the 5′ end Cluc tilingGAGATCACAGGCTCCTTCAGCATCAAAAGA 1380 Note that the Cas13a spacers 49Dguide 18 (SEQ ID NO: 450) are truncated by two nucleotides at the 5′ endCluc tiling GCTTTGACCGGCGAAGAGACTATTGCAGAG 1461Note that the Cas13a spacers 49D guide 19 (SEQ ID NO: 451)are truncated by two nucleotides at the 5′ end Cluc tilingGCCCCTCAGGCAATACTCGTACATGCATCG 1539 Note that the Cas13a spacers 49Dguide 20 (SEQ ID NO: 452) are truncated by two nucleotides at the 5′ endCluc tiling GCTGGTACTTCTAGGGTGTCTCCATGCTTT 1619Note that the Cas13a spacers 49D guide 21 (SEQ ID NO: 453)are truncated by two nucleotides at the 5′ end Non-targetingGGTAATGCCTGGCTTGTCGACGCATAGTCTG N/A Note that the Cas13a spacers 49Dguide 1 (SEQ ID NO: 454) are truncated by two nucleotides at the 5′ endNon-targeting GGGAACCTTGGCCGTTATAAAGTCTGACCAG N/ANote that the Cas13a spacers 49D guide 2 (SEQ ID NO: 455)are truncated by two nucleotides at the 5′ end Non-targetingGGAGGGTGAGAATTTAGAACCAAGATTGTTG N/A Note that the Cas13a spacers 49Dguide 3 (SEQ ID NO: 456) are truncated by two nucleotides at the 5′ end

TABLE 21Guide sequences used in this study for RNA editing in mammalian cells.Mismatched base flips are capitalized First Name Spacer sequence NotesFIG. Tiling 30 nt 30 gCatcctgcggcctctactctgcattcaatt Has a 5′ G for 50Cmismatch distance (SEQ ID NO: 457) U6 expression Tiling 30 nt 28gacCatcctgcggcctctactctgcattcaa Has a 5′ G for 50C mismatch distance(SEQ ID NO: 458) U6 expression Tiling 30 nt 26gaaaCatcctgcggcctctactctgcattc Has a 5′ G for 50C mismatch distance(SEQ ID NO: 459) U6 expression Tiling 30 nt 24gctaaacCatcctgcggcctctactctgcat Has a 5′ G for 50C mismatch distance(SEQ ID NO: 460) U6 expression Tiling 30 nt 22gttctaaacCatcctgcggcctctactctgc Has a 5′ G for 50C mismatch distance(SEQ ID NO: 461) U6 expression Tiling 30 nt 20gtgttctaaacCatcctgcggcctctactct Has a 5′ G for 50C mismatch distance(SEQ ID NO: 462) U6 expression Tiling 30 nt 18gaatgttctaaacCatcctgcggcctctact Has a 5′ G for 50C mismatch distance(SEQ ID NO: 463) U6 expression Tiling 30 nt 16gagaatgttctaaacCatcctgcggcctcta Has a 5′ G for 50C mismatch distance(SEQ ID NO: 464) U6 expression Tiling 30 nt 14gatagaatgttctaaacCatcctgcggcctc Has a 5′ G for 50C mismatch distance(SEQ ID NO: 465) U6 expression Tiling 30 nt 12gccatagaatgttctaaacCatcctgcggcc Has a 5′ G for 50C mismatch distance(SEQ ID NO: 466) U6 expression Tiling 30 nt 10gttccatagaatgttctaaacCatcctgcgg Has a 5′ G for 50C mismatch distance(SEQ ID NO: 467) U6 expression Tiling 30 nt 8gctttccatagaatgttctaaacCatcctgc Has a 5′ G for 50C mismatch distance(SEQ ID NO: 468) U6 expression Tiling 30 nt 6gctctttccatagaatgttctaaacCatcct Has a 5′ G for 50C mismatch distance(SEQ ID NO: 469) U6 expression Tiling 30 nt 4gatctctttccatagaatgttctaaacCatc Has a 5′ G for 50C mismatch distance(SEQ ID NO: 470) U6 expression Tiling 30 nt 2ggaatctctttccatagaatgttctaaacCa Has a 5′ G for 50C mismatch distance(SEQ ID NO: 471) U6 expression Tiling 50 nt 50gCatcctgcggcctctactctgcattcaattacatactgacacattcggca Has a 5′ G for 50Cmismatch distance (SEQ ID NO: 472) U6 expression Tiling 50 nt 48gacCatcctgcggcctctactctgcattcaattacatactgacacattcgg Has a 5′ G for 50Cmismatch distance (SEQ ID NO: 473) U6 expression Tiling 50 nt 46gaaacCatcctgcggcctctactctgcattcaattacatactgacacattc Has a 5′ G for 50Cmismatch distance (SEQ ID NO: 474) U6 expression Tiling 50 nt 44gctaaacCatcctgcggcctctactctgcattcaattacatactgacacat Has a 5′ G for 50Cmismatch distance (SEQ ID NO: 475) U6 expression Tiling 50 nt 42gttctaaacCatcctgcggcctctactctgcattcaattacatactgacac Has a 5′ G for 50Cmismatch distance (SEQ ID NO: 476) U6 expression Tiling 50 nt 40gtgttctaaacCatcctgcggcctctactctgcattcaattacatactgac Has a 5′ G for 50Cmismatch distance (SEQ ID NO: 477) U6 expression Tiling 50 nt 38gaatgttctaaacCatcctgcggcctctactctgcattcaattacatactg Has a 5′ G for 50Cmismatch distance (SEQ ID NO: 478) U6 expression Tiling 50 nt 36gagaatgttctaaacCatcctgcggcctctactctgcattcaattacatac Has a 5′ G for 50Cmismatch distance (SEQ ID NO: 479) U6 expression Tiling 50 nt 34gatagaatgttctaaacCatcctgcggcctctactctgcattcaattacat Has a 5′ G for 50Cmismatch distance (SEQ ID NO: 480) U6 expression Tiling 50 nt 32gccatagaatgttctaaacCatcctgcggcctctactctgcattcaattac Has a 5′ G for 50Cmismatch distance (SEQ ID NO: 481) U6 expression Tiling 50 nt 30gttccatagaatgttctaaacCatcctgcggcctctactctgcattcaatt Has a 5′ G for 50Cmismatch distance (SEQ ID NO: 482) U6 expression Tiling 50 nt 28gctttccatagaatgttctaaacCatcctgcggcctctactctgcattcaa Has a 5′ G for 50Cmismatch distance (SEQ ID NO: 483) U6 expression Tiling 50 nt 26gctctttccatagaatgttctaaacCatcctgcggcctctactctgcattc Has a 5′ G for 50Cmismatch distance (SEQ ID NO: 484) U6 expression Tiling 50 nt 24gatctctttccatagaatgttctaaacCatcctgcggcctctactctgcat Has a 5′ G for 50Cmismatch distance (SEQ ID NO: 485) U6 expression Tiling 50 nt 22ggaatctctttccatagaatgttctaaacCatcctgcggcctctactctgc Has a 5′ G for 50Cmismatch distance (SEQ ID NO: 486) U6 expression Tiling 50 nt 20gtggaatctctttccatagaatgttctaaacCatcctgcggcctctactct Has a 5′ G for 50Cmismatch distance (SEQ ID NO: 487) U6 expression Tiling 50 nt 18gactggaatctctttccatagaatgttctaaacCatcctgcggcctctact Has a 5′ G for 50Cmismatch distance (SEQ ID NO: 488) U6 expression Tiling 50 nt 16ggaactggaatctctttccatagaatgttctaaacCatcctgcggcctcta Has a 5′ G for 50Cmismatch distance (SEQ ID NO: 489) U6 expression Tiling 50 nt 14gtggaactggaatctctttccatagaatgttctaaacCatcctgcggccta Has a 5′ G for 50Cmismatch distance (SEQ ID NO: 490) U6 expression Tiling 50 nt 12gcctggaactggaatctctttccatagaatgttctaaacCatcctgcggcc Has a 5′ G for 50Cmismatch distance (SEQ ID NO: 491) U6 expression Tiling 50 nt 10gttcctggaactggaatctctttccatagaatgttctaaacCatcctgcgg Has a 5′ G for 50Cmismatch distance (SEQ ID NO: 492) U6 expression Tiling 50 nt 8gggttcctggaactggaatctctttccatagaatgttctaaacCatcctgc Has a 5′ G for 50Cmismatch distance (SEQ ID NO: 493) U6 expression Tiling 50 nt 6gcaggttcctggaactggaatctctttccatagaatgttctaaacCatcct Has a 5′ G for 50Cmismatch distance (SEQ ID NO: 494) U6 expression Tiling 50 nt 4gaccaggttcctggaactggaatctctttccatagaatgttctaaacCatc Has a 5′ G for 50Cmismatch distance (SEQ ID NO: 495) U6 expression Tiling 50 nt 2ggtaccaggttcctggaactggaatctctttccatagaatgttctaaacCa Has a 5′ G for 50Cmismatch distance (SEQ ID NO: 496) U6 expression Tiling 70 nt 70gCatcctgcggcctctactctgcattcaattacatactgacacattcggca Has a 5′ G for 50Cmismatch distance acatgtttttcctggtttat U6 expression (SEQ ID NO: 497)Tiling 70 nt 68 gacCatcctgcggcctctactctgcattcaattacatactgacacattcggHas a 5′ G for 50C mismatch distance caacatgtttttcctggttt U6 expression(SEQ ID NO: 498) Tiling 70 nt 66gaaacCatcctgcggcctctactctgcattcaattacatactgacacattc Has a 5′ G for 50Cmismatch distance ggcaacatgtttttcctggt U6 expression (SEQ ID NO: 499)Tiling 70 nt 64 gctaaacCatcctgcggcctctactctgcattcaattacatactgacacatHas a 5′ G for 50C mismatch distance tcggcaacatgtttttcctg U6 expression(SEQ ID NO: 500) Tiling 70 nt 62gttctaaacCatcctgcggcctctactctgcattcaattacatactgacac Has a 5′ G for 50Cmismatch distance attcggcaacatgtttttcc U6 expression (SEQ ID NO: 501)Tiling 70 nt 60 gtgttctaaacCatcctgcggcctctactctgcattcaattacatactgacHas a 5′ G for 50C mismatch distance acattcggcaacatgttttt U6 expression(SEQ ID NO: 502) Tiling 70 nt 58gaatgttctaaacCatcctgcggcctctactctgcattcaattacatactg Has a 5′ G for 50Cmismatch distance acacattcggcaacatgttt U6 expression (SEQ ID NO: 503)Tiling 70 nt 56 gagaatgttctaaacCatcctgcggcctctactctgcattcaattacatacHas a 5′ G for 50C mismatch distance tgacacattcggcaacatgt U6 expression(SEQ ID NO: 504) Tiling 70 nt 54gatagaatgttctaaacCatcctgcggcctctactctgcattcaattacat Has a 5′ G for 50Cmismatch distance actgacacattcggcaacat U6 expression (SEQ ID NO: 505)Tiling 70 nt 52 gccatagaatgttctaaacCatcctgcggcctctactctgcattcaattacHas a 5′ G for 50C mismatch distance atactgacacattcggcaac U6 expression(SEQ ID NO: 506) Tiling 70 nt 50gttccatagaatgttctaaacCatcctgcggcctctactctgcattcaatt Has a 5′ G for 50Cmismatch distance acatactgacacattcggca U6 expression (SEQ ID NO: 507)Tiling 70 nt 48 gctttccatagaatgttctaaacCatcctgcggcctctactctgcattcaaHas a 5′ G for 50C mismatch distance ttacatactgacacattcgg U6 expression(SEQ ID NO: 508) Tiling 70 nt 46gctctttccatagaatgttctaaacCatcctgcggcctctactctgcattc Has a 5′ G for 50Cmismatch distance aattacatactgacacattc U6 expression (SEQ ID NO: 509)Tiling 70 nt 44 gatctctttccatagaatgttctaaacCatcctgcggcctctactctgcatHas a 5′ G for 50C mismatch distance tcaattacatactgacacat U6 expression(SEQ ID NO: 510) Tiling 70 nt 42ggaatctctttccatagaatgttctaaacCatcctgcggcctctactctgc Has a 5′ G for 50Cmismatch distance attcaattacatactgacac U6 expression (SEQ ID NO: 511)Tiling 70 nt 40 gtggaatctctttccatagaatgttctaaacCatcctgcggcctctactctHas a 5′ G for 50C mismatch distance gcattcaattacatactgac U6 expression(SEQ ID NO: 512) Tiling 70 nt 38gactggaatctctttccataggaatgttctaaacCatcctgcggcctctac Has a 5′ G for 50Cmismatch distance tctgcattcaattacatactg U6 expression (SEQ ID NO: 513)Tiling 70 nt 36 ggaactggaatctctttccatagaatgttctaaacCatcctgcggcctctaHas a 5′ G for 50C mismatch distance ctctgcattcaattacatac U6 expression(SEQ ID NO: 514) Tiling 70 nt 34gtggaactggaatctctttccatagaatgttctaaacCatcctgcggcctc Has a 5′ G for 50Cmismatch distance tactctgcattcaattacat U6 expression (SEQ ID NO: 515)Tiling 70 nt 32 gcctggaactggaatctctttccatagaatgttctaaacCatcctgcggccHas a 5′ G for 50C mismatch distance tctactctgcattcaattac U6 expression(SEQ ID NO: 516) Tiling 70 nt 30gttcctggaactggaatctctttccatagaatgttctaaacCatcctgcgg Has a 5′ G for 50Cmismatch distance cctctactctgcattcaatt U6 expression (SEQ ID NO: 517)Tiling 70 nt 28 gggttcctggaactggaatctctttccatagaatgttctaaacCatcctgcHas a 5′ G for 50C mismatch distance ggcctctactctgcattcaa U6 expression(SEQ ID NO: 518) Tiling 70 nt 26gcaggttcctggaactggaatctctttccatagaatgttctaaacCatcct Has a 5′ G for 50Cmismatch distance gcggcctctactctgcattc U6 expression (SEQ ID NO: 519)Tiling 70 nt 24 gaccaggttcctggaactggaatctctttccatagaatgttctaaacCatcHas a 5′ G for 50C mismatch distance ctgcggcctctactctgcat U6 expression(SEQ ID NO: 520) Tiling 70 nt 22ggtaccaggttcctggaactggaatctctttccatagaatgttctaaacCa Has a 5′ G for 50Cmismatch distance tcctgcggcctctactctgc U6 expression (SEQ ID NO: 521)Tiling 70 nt 20 gatgtaccaggttcctggaactggaatctctttccatagaatgttctaaacHas a 5′ G for 50C mismatch distance Catcctgcggcctctactct U6 expression(SEQ ID NO: 522) Tiling 70 nt 18ggtatgtaccaggttcctggaactggaatctctttccatagaatgttctaa Has a 5′ G for 50Cmismatch distance acCatcctgcggcctctact U6 expression (SEQ ID NO: 523)Tiling 70 nt 16 gacgtatgtaccaggttcctggaactggaatctctttccatagaatgttctHas a 5′ G for 50C mismatch distance aaacCatcctgcggcctcta U6 expression(SEQ ID NO: 524) Tiling 70 nt 14gacacgtatgtaccaggttcctggaactggaatctctttccatagaatgtt Has a 5′ G for 50Cmismatch distance ctaaacCatcctgcggcctc U6 expression (SEQ ID NO: 525)Tiling 70 nt 12 gcaacacgtatgtaccaggttcctggaactggaatctctttccatagaatgHas a 5′ G for 50C mismatch distance ttctaaacCatcctgcggcc U6 expression(SEQ ID NO: 526) Tiling 70 nt 10gcccaacacgtatgtaccaggttcctggaactggaatctctttccatagaa Has a 5′ G for 50Cmismatch distance tgttctaaacCatcctgcgg U6 expression (SEQ ID NO: 527)Tiling 70 nt 8 ggacccaacacgtatgtaccaggttcctggaactggaatctctttccatagHas a 5′ G for 50C mismatch distance aatgttctaaacCatcctgc U6 expression(SEQ ID NO: 528) Tiling 70 nt 6gttgacccaacacgtatgtaccaggttcctggaactggaatctctttccat Has a 5′ G for 50Cmismatch distance agaatgttctaaacCatcct U6 expression (SEQ ID NO: 529)Tiling 70 nt 4 gccttgacccaacacgtatgtaccaggttcctggaactggaatctctttccHas a 5′ G for 50C mismatch distance atagaatgttctaaacCatc U6 expression(SEQ ID NO: 530) Tiling 70 nt 2gttccttgacccaacacgtatgtaccaggttcctggaactggaatctcttt Has a 5′ G for 50Cmismatch distance ccatagaatgttctaaacCa U6 expression (SEQ ID NO: 531)Tiling 84 nt 84 gCatcctgcggcctctactctgcattcaattacatactgacacattcggcaHas a 5′ G for 50C mismatch distance acatgtttttcctggtttattttcacacagtccaU6 expression (SEQ ID NO: 532) Tiling 84 nt 82gacCatcctgcggcctctactctgcattcaattacatactgacacattcgg Has a 5′ G for 50Cmismatch distance caacatgtttttcctggtttattttcacacagtc U6 expression(SEQ ID NO: 533) Tiling 84 nt 80gaaacCatcctgcggcctctactctgcattcaattacatactgacacattc Has a 5′ G for 50Cmismatch distance ggcaacatgtttttcctggtttattttcacacag U6 expression(SEQ ID NO: 534) Tiling 84 nt 78gctaaacCatcctgcggcctctactctgcattcaattacatactgacacat Has a 5′ G for 50Cmismatch distance tcggcaacatgtttttcctggtttattttcacac U6 expression(SEQ ID NO: 535) Tiling 84 nt 76gttctaaacCatcctgcggcctctactctgcattcaattacatactgacac Has a 5′ G for 50Cmismatch distance attcggcaacatgtttttcctggtttattttcac U6 expression(SEQ ID NO: 536) Tiling 84 nt 74gtgttctaaacCatcctgcggcctctactctgcattcaattacatactgac Has a 5′ G for 50Cmismatch distance acattcggcaacatgtttttcctggtttattttc U6 expression(SEQ ID NO: 537) Tiling 84 nt 72gaatgttctaaacCatcctgcggcctctactctgcattcaattacatactg Has a 5′ G for 50Cmismatch distance acacattcggcaacatgtttttcctggtttattt U6 expression(SEQ ID NO: 538) Tiling 84 nt 70gagaatgttctaaacCatcctgcggcctctactctgcattcaattacatac Has a 5′ G for 50Cmismatch distance tgacacattcggcaacatgtttttcctggtttat U6 expression(SEQ ID NO: 539) Tiling 84 nt 68gatagaatgttctaaacCatcctgcggcctctactctgcattcaattacat Has a 5′ G for 50Cmismatch distance actgacacattcggcaacatgtttttcctggttt U6 expression(SEQ ID NO: 540) Tiling 84 nt 66gccatagaatgttctaaacCatcctgcggcctctactctgcattcaattac Has a 5′ G for 50Cmismatch distance atactgacacattcggcaacatgtttttcctggt U6 expression(SEQ ID NO: 541) Tiling 84 nt 64gttccatagaatgttctaaacCatcctgcggcctctactctgcattcaatt Has a 5′ G for 50Cmismatch distance acatactgacacattcggcaacatgtttttcctg U6 expression(SEQ ID NO: 542) Tiling 84 nt 62gctttccatagaatgttctaaacCatcctgcggcctctactctgcattcaa Has a 5′ G for 50Cmismatch distance ttacatactgacacattcggcaacatgtttttcc U6 expression(SEQ ID NO: 543) Tiling 84 nt 60gctctttccatagaatgttctaaacCatcctgcggcctctactctgcattc Has a 5′ G for 50Cmismatch distance aattacatactgacacattcggcaacatgttttt U6 expression(SEQ ID NO: 544) Tiling 84 nt 58gatctctttccatagaatgttctaaacCatcctgcggcctctactctgcat Has a 5′ G for 50Cmismatch distance tcaattacatactgacacattcggcaacatgttt U6 expression(SEQ ID NO: 545) Tiling 84 nt 56ggaatctctttccatagaatgttctaaacCatcctgcggcctctactctgc Has a 5′ G for 50Cmismatch distance attcaattacatactgacacattcggcaacatgt U6 expression(SEQ ID NO: 546) Tiling 84 nt 54gtggaatctctttccatagaatgttctaaacCatcctgcggcctctactct Has a 5′ G for 50Cmismatch distance gcattcaattacatactgacacattcggcaacat U6 expression(SEQ ID NO: 547) Tiling 84 nt 52gactggaatctctttccatagaatgttctaaacCatcctgcggcctctact Has a 5′ G for 50Cmismatch distance ctgcattcaattacatactgacacattcggcaac U6 expression(SEQ ID NO: 548) Tiling 84 nt 50ggaactggaatctctttccatagaatgttctaaacCatcctgcggcctcta Has a 5′ G for 50Cmismatch distance ctctgcattcaattacatactgacacattcggca U6 expression(SEQ ID NO: 549) Tiling 84 nt 48gtggaactggaatctctttccatagaatgttctaaacCatcctgcggcctc Has a 5′ G for 50Cmismatch distance tactctgcattcaattacatactgacacattcgg U6 expression(SEQ ID NO: 550) Tiling 84 nt 46gcctggaactggaatctctttccatagaatgttctaaacCatcctgcggcc Has a 5′ G for 50Cmismatch distance tctactctgcattcaattacatactgacacattc U6 expression(SEQ ID NO: 551) Tiling 84 nt 44gttcctggaactggaatctctttccatagaatgttctaaacCatcctgcgg Has a 5′ G for 50Cmismatch distance cctctactctgcattcaattacatactgacacat U6 expression(SEQ ID NO: 552) Tiling 84 nt 42gggttcctggaactggaatctctttccatagaatgttctaaacCatcctgc Has a 5′ G for 50Cmismatch distance ggcctctactctgcattcaattacatactgacac U6 expression(SEQ ID NO: 553) Tiling 84 nt 40gcaggttcctggaactggaatctctttccatagaatgttctaaacCatcct Has a 5′ G for 50Cmismatch distance gcggcctctactctgcattcaattacatactgac U6 expression(SEQ ID NO: 554) Tiling 84 nt 38gaccaggttcctggaactggaatctctttccatagaatgttctaaacCatc Has a 5′ G for 50Cmismatch distance ctgcggcctctactctgcattcaattacatactg U6 expression(SEQ ID NO: 555) Tiling 84 nt 35ggtaccaggttcctggaactggaatctctttccatagaatgttctaaacCa Has a 5′ G for 50Cmismatch distance tcctgcggcctctactctgcattcaattacatac U6 expression(SEQ ID NO: 556) Tiling 84 nt 34gatgtaccaggttcctggaactggaatctctttccatagaatgttctaaac Has a 5′ G for 50Cmismatch distance Catcctgcggcctctactctgcattcaattacat U6 expression(SEQ ID NO: 557) Tiling 84 nt 32ggtatgtaccaggttcctggaactggaatctctttccatagaatgttctaa Has a 5′ G for 50Cmismatch distance acCatcctgcggcctctactctgcattcaattac U6 expression(SEQ ID NO: 558) Tiling 84 nt 30gacgtatgtaccaggttcctggaactggaatctctttccatagaatgttct Has a 5′ G for 50Cmismatch distance aaacCatcctgcggcctctactctgcattcaatt U6 expression(SEQ ID NO: 559) Tiling 84 nt 28gacacgtatgtaccaggttcctggaactggaatctctttccatagaatgtt Has a 5′ G for 50Cmismatch distance ctaaacCatcctgcggcctctactctgcattcaa U6 expression(SEQ ID NO: 560) Tiling 84 nt 26gcaacacgtatgtaccaggttcctggaactggaatctctttccatagaatg Has a 5′ G for 50Cmismatch distance ttctaaacCatcctgcggcctctactctgcattc U6 expression(SEQ ID NO: 561) Tiling 84 nt 24gcccaacacgtatgtaccaggttcctggaactggaatctctttccatagaa Has a 5′ G for 50Cmismatch distance tgttctaaacCatcctgcggcctctactctgcat U6 expression(SEQ ID NO: 562) Tiling 84 nt 22ggacccaacacgtatgtaccaggttcctggaactggaatctctttccatag Has a 5′ G for 50Cmismatch distance aatgttctaaacCatcctgcggcctctactctgc U6 expression(SEQ ID NO: 563) Tiling 84 nt 20gttgacccaacacgtatgtaccaggttcctggaactggaatctctttccat Has a 5′ G for 50Cmismatch distance agaatgttctaaacCatcctgcggcctctactct U6 expression(SEQ ID NO: 564) Tiling 84 nt 18gccttgacccaacacgtatgtaccaggttcctggaactggaatctctttcc Has a 5′ G for 50Cmismatch distance atagaatgttctaaacCatcctgcggcctctact U6 expression(SEQ ID NO: 565) Tiling 84 nt 16gttccttgacccaacacgtatgtaccaggttcctggaactggaatctcttt Has a 5′ G for 50Cmismatch distance ccatagaatgttctaaacCatcctgcggcctcta U6 expression(SEQ ID NO: 566) Tiling 84 nt 14gggttccttgacccaacacgtatgtaccaggttcctggaactggaatctct Has a 5′ G for 50Cmismatch distance ttccatagaatgttctaaacCatcctgcggcctc U6 expression(SEQ ID NO: 567) Tiling 84 nt 12gttggttccttgacccaacacgtatgtaccaggttcctggaactggaatct Has a 5′ G for 50Cmismatch distance ctttccatagaatgttctaaacCatcctgcggcc U6 expression(SEQ ID NO: 568) Tiling 84 nt 10gccttggttccttgacccaacacgtatgtaccaggttcctggaactggaat Has a 5′ G for 50Cmismatch distance ctctttccatagaatgttctaaacCatcctgcgg U6 expression(SEQ ID NO: 569) Tiling 84 nt 8ggcccttggttccttgacccaacacgtatgtaccaggttcctggaactgga Has a 5′ G for 50Cmismatch distance atctctttccatagaatgttctaaacCatcctgc U6 expression(SEQ ID NO: 570) Tiling 84 nt 6gccgcccttggttccttgacccaacacgtatgtaccaggttcctggaactg Has a 5′ G for 50Cmismatch distance gaatctctttccatagaatgttctaaacCatcct U6 expression(SEQ ID NO: 571) Tiling 84 nt 4gcgccgcccttggttccttgacccaacacgtatgtaccaggttcctggaac Has a 5′ G for 50Cmismatch distance tggaatctctttccatagaatgttctaaacCatc U6 expression(SEQ ID NO: 572) Tiling 84 nt 2ggtcgccgcccttggttccttgacccaacacgtatgtaccaggttcctgga Has a 5′ G for 50Cmismatch distance actggaatctctttccatagaatgttctaaacCa U6 expression(SEQ ID NO: 573) ADAR non- GTAATGCCTGGCTTGTCGACGCATAGTCTG Has a 5′ G for50C targeting guide (SEQ ID NO: 574) U6 expression PFS bindinggaaaacgcaggttcctcCagtttcgggagcagcgcacgtctccctgtagtc Has a 5′ G for 51Bscreen guide for (SEQ ID NO: 575) U6 expression TAG motif PFS bindinggacgcaggttcctctagCttcgggagcagcgcacgtctccctgtagtcaag Has a 5′ G for 51Bscreen guide for (SEQ ID NO: 576) U6 expression TAG motif PFS bindingGTAATGCCTGGCTTGTCGACGCATAGTCTG Has a 5′ G for 51B screen non-(SEQ ID NO: 577) U6 expression targeting Motif preferencegatagaatgttctaaacCatcctgcggcctctactctgcattcaattacat Has a 5′ G for 51Ctargeting guide (SEQ ID NO: 578) U6 expression Motif preferenceGTAATGCCTGGCTTGTCGACGCATAGTCTG Has a 5′ G for 51C non-targeting(SEQ ID NO: 579) U6 expression guide PPIB tilinggCaaggccacaaaattatccactgtttttggaacagtctttccgaagagac Has a 5′ G for 57Dguide 50 mismatch (SEQ ID NO: 580) U6 expression distance PPIB tilinggcctgtagcCaaggccacaaaattatccactgtttttggaacagtctttcc Has a 5′ G for 57Dguide 42 mismatch (SEQ ID NO: 581) U6 expression distance PPIB tilinggctttctctcctgtagcCaaggccacaaaattatccactgtttttggaaca Has a 5′ G for 57Dguide 34 mismatch (SEQ ID NO: 582) U6 expression distance PPIB tilingggccaaatcctttctctcctgtagcCaaggccacaaaattatccactgttt Has a 5′ G for 57Dguide 26 mismatch (SEQ ID NO: 583) U6 expression distance PPIB tilinggtttttgtagccaaatcctttctctcctgtagcCaaggccacaaaattatc Has a 5′ G for 57Dguide 18 mismatch (SEQ ID NO: 584) U6 expression distance PPIB tilinggatttgctgtttttgtagccaaatcctttctctcctgtagcCaaggccaca Has a 5′ G for 57Dguide 10 mismatch (SEQ ID NO: 585) U6 expression distance PPIB tilinggacgatggaatttgctgtttttgtagccaaatcctttctctcctgtagcCa Has a 5′ G for 57Dguide 2 mismatch (SEQ ID NO: 586) U6 expression distanceTargeting guide, gatagaatgttctaaacGatcctgcggcctctactctgcattcaattacatHas a 5′ G for 57D opposite base G (SEQ ID NO: 587) U6 expressionTargeting guide, gatagaatgttctaaacAatcctgcggcctctactctgcattcaattacatHas a 5′ G for 57D opposite base A (SEQ ID NO: 588) U6 expressionTargeting guide, gatagaatgttctaaacTatcctgcggcctctactctgcattcaattacatHas a 5′ G for 57D opposite base C (SEQ ID NO: 589) U6 expressionAVPR2 guide 37 ggtcccacgcggccCacagctgcaccaggaagaagggtgcccagcacagcaHas a 5′ G for 52A mismatch distance (SEQ ID NO: 590) U6 expressionAVPR2 guide 35 ggggtcccacgcggccCacagctgcaccaggaagaagggtgcccagcacagHas a 5′ G for 52A mismatch distance (SEQ ID NO: 591) U6 expressionAVPR2 guide 33 gccgggtcccacgcggccCacagctgcaccaggaagaagggtgcccagcacHas a 5′ G for 52A mismatch distance (SEQ ID NO: 592) U6 expressionFANCC guide 37 gggtgatgacatccCaggcgatcgtgtggcctccaggagcccagagcaggaHas a 5′ G for 52B mismatch distance (SEQ ID NO: 593) U6 expressionFANCC guide 35 gagggtgatgacatccCaggcgatcgtgtggcctccaggagcccagagcagHas a 5′ G for 52B mismatch distance (SEQ ID NO: 594) U6 expressionFANCC guide 32 gatcagggtgatgacatccCaggcgatcgtgtggcctccaggagcccagagHas a 5′ G for 52B mismatch distance (SEQ ID NO: 595) U6 expressionSynthetic disease ggtggctccattcactcCaatgctgagcacttccacagagtgggttaaagcHas a 5′ G for 52E gene target IL2RG (SEQ ID NO: 596) U6 expressionSynthetic disease gtttctaatatattttgCcagactgatggactattctcaattaataatgatHas a 5′ G for 52E gene target F8 (SEQ ID NO: 597) U6 expressionSynthetic disease gagatgttgctgtggatCcagtccacagccagcccgtcgggggcctggatgHas a 5′ G for 52E gene target LDLR (SEQ ID NO: 598) U6 expressionSynthetic disease gcaggccggcccagctgCcaggtgcacctgctcggagcatcgggccggatcHas a 5′ G for 52E gene target CBS (SEQ ID NO: 599) U6 expressionSynthetic disease gcaaagaacctctgggtCcaagggtagaccaccagcagcctgcccagggccHas a 5′ G for 52E gene target HBB (SEQ ID NO: 600) U6 expressionSynthetic disease gaagagaaacttagtttCcagggctttggtagagggcaaaggttgatagcaHas a 5′ G for 52E gene target ALDOB (SEQ ID NO: 601) U6 expressionSynthetic disease gtcagcctagtgcagagCcactggtagttggtggttagagtttcaagttccHas a 5′ G for 52E gene target DMD (SEQ ID NO: 602) U6 expressionSynthetic disease ggctcattgtgaacaggCcagtaatgtccgggatggggcggcataggcgggHas a 5′ G for 52E gene target SMAD4 (SEQ ID NO: 603) U6 expressionSynthetic disease gtagctaaagaacttgaCcaagacatatcaggatccacctcagctcctagaHas a 5′ G for 52E gene target BRCA2 (SEQ ID NO: 604) U6 expressionSynthetic disease ggggcattgttctgtgcCcagtcctgctggtagacctgctccccggtggctHas a 5′ G for 52E gene target (SEQ ID NO: 605) U6 expression GRIN2ASynthetic disease gagaagtcgttcatgtgCcaccgtgggagcgtacagtcatcattgatcttgHas a 5′ G for 52E gene target SCN9A (SEQ ID NO: 606) U6 expressionSynthetic disease gggattaatgctgaacgCaccaaagttcatcccaccacccatattactaccHas a 5′ G for 52E gene target  (SEQ ID NO: 607) U6 expression TARDBPSynthetic disease gctccaaaggctttcctCcactgttgcaaagttattgaatcccaagacacaHas a 5′ G for 52E gene target CFTR (SEQ ID NO: 608) U6 expressionSynthetic disease gatgaatgaacgatttcCcagaactccctaatcagaacagagtccctggtaHas a 5′ G for 52E gene target UBE3A (SEQ ID NO: 609) U6 expressionSynthetic disease ggagcctctgccggagcCcagagaacccgagagtcagacagagccagcgccHas a 5′ G for 52E gene target SMPD1 (SEQ ID NO: 610) U6 expressionSynthetic disease ggcttccgtggagacacCcaatcaatttgaagagatcttgaagtgatgccaHas a 5′ G for 52E gene target USH2A (SEQ ID NO: 611) U6 expressionSynthetic disease gtgggactgccctcctcCcatttgcagatgccgtcgtagaatcgcagcaggHas a 5′ G for 52E gene target MEN1 (SEQ ID NO: 612) U6 expressionSynthetic disease gcttcttcaatagttctCcagctacactggcaggcatatgcccgtgttcctHas a 5′ G for 52E gene target (SEQ ID NO: 613) U6 expression C8orf37Synthetic disease gattccttttcttcgtcCcaattcacctcagtggctagtcgaagaatgaagHas a 5′ G for 52E gene target MLH1 (SEQ ID NO: 614) U6 expressionSynthetic disease gcagcttcagcaccttcCagtcagactcctgcttcaagcactgcagcaggaHas a 5′ G for 52E gene target TSC2 (SEQ ID NO: 615) U6 expressionSynthetic disease gccatttgcttgcagtgCcactccagaggattccggattgccataaatactHas a 5′ G for 52E gene target NF1 (SEQ ID NO: 616) U6 expressionSynthetic disease gttcaatagttttggtcCagtatcgtttacagcccttcttggtagatttcaHas a 5′ G for 52E gene target MSH2 (SEQ ID NO: 617) U6 expressionSynthetic disease ggcaaccgtcttctgacCaaatggcagaacatttgtccccaactttccactHas a 5′ G for 52E gene target SMN1 (SEQ ID NO: 618) U6 expressionSynthetic disease gcgactttccaatgaacCactgaagcccaggtatgacaaagccgatgatctHas a 5′ G for 52E gene target (SEQ ID NO: 619) U6 expression SH3TC2Synthetic disease gtttacactcatgcttcCacagctttaacagatcatttggttccttgatgaHas a 5′ G for 52E gene target DNAH5 (SEQ ID NO: 620) U6 expressionSynthetic disease gcttaagcttccgtgtcCagccttcaggcagggtggggtcatcatacatggHas a 5′ G for 52E gene target MECP2 (SEQ ID NO: 621) U6 expressionSynthetic disease ggacagctgggctgatcCatgatgtcatccagaaacactggggaccctcagHas a 5′ G for 52E gene target (SEQ ID NO: 622) U6 expression ADGRV1Synthetic disease gtctcatctcaactttcCatatccgtatcatggaatcatagcatcctgtaaHas a 5′ G for 52E gene target AHI1 (SEQ ID NO: 623) U6 expressionSynthetic disease gcatgcagacgcggttcCactcgcagccacagttccagcaccactcgagccHas a 5′ G for 52E gene target PRKN (SEQ ID NO: 624) U6 expressionSynthetic disease gttggttagggtcaaccCagtattctccactcttgagttcaggatggcagaHas a 5′ G for 52E gene target (SEQ ID NO: 625) U6 expression COL3A1Synthetic disease gctacactgtccaacacCcactctcgggtcaccacaggtgcctcacacatcHas a 5′ G for 52E gene target BRCA1 (SEQ ID NO: 626) U6 expressionSynthetic disease gctgcactgtgtaccccCagagctccgtgttgccgacatcctggggtggctHas a 5′ G for 52E gene target (SEQ ID NO: 627) U6 expression MYBPC3Synthetic disease gagcttcctgccactccCaacaggtttcacagtaagcgcgtatctgttccaHas a 5′ G for 52E gene target APC (SEQ ID NO: 628) U6 expressionSynthetic disease gacggcaagagcttaccCagtcacttgtgtggagacttaaatacttgcataHas a 5′ G for 52E gene target BMPR2 (SEQ ID NO: 629) U6 expressionKRAS tiling guide gCaaggccacaaaattatccactgtttttggaacagtctttccgaagagacHas a 5′ G for 53A 50 mismatch (SEQ ID NO: 630) U6 expression distanceKRAS tiling guide gcctgtagcCaaggccacaaaattatccactgtttttggaacagtctttccHas a 5′ G for 53A 42 mismatch (SEQ ID NO: 631) U6 expression distanceKRAS tiling guide gctttctctcctgtagcCaaggccacaaaattatccactgtttttggaacaHas a 5′ G for 53A 34 mismatch (SEQ ID NO: 632) U6 expression distanceKRAS tiling guide ggccaaatcctttctctcctgtagcCaaggccacaaaattatccactgtttHas a 5′ G for 53A 26 mismatch (SEQ ID NO: 633) U6 expression distanceKRAS tiling guide gtttttgtagccaaatcctttctctcctgtagcCaaggccacaaaattatcHas a 5′ G for 53A 18 mismatch (SEQ ID NO: 634) U6 expression distanceKRAS tiling guide gatttgctgtttttgtagccaaatcctttctctcctgtagcCaaggccacaHas a 5′ G for 53A 10 mismatch (SEQ ID NO: 635) U6 expression distanceKRAS tiling guide gacgatggaatttgctgtttttgtagccaaatcctttctctcctgtagcCaHas a 5′ G for 53A 2 mismatch (SEQ ID NO: 636) U6 expression distanceKRAS tiling non- GTAATGCCTGGCTTGTCGACGCATAGTCTG Has a 5′ G for 53Atargeting guide (SEQ ID NO: 637) U6 expression Luciferase W85XgatagaatgttctaaacCatcctgcggcctctactctgcattcaattacat Has a 5′ G for 53Btargeting guide (SEQ ID NO: 638) U6 expression for transcriptomespecificity Non-targeting GCAGGGTTTTCCCAGTCACGACGTTGTAAAGTTGHas a 5′ G for 53C guide for (SEQ ID NO: 639) U6 expressiontranscriptome specificity endogenous KRASgtcaaggcactcttgccCacgccaccagctccaactaccacaagtttatat Has a 5′ G for 54Fguide 2 (SEQ ID NO: 640) U6 expression endogenous PPIBgcaaagatcacccggccCacatcttcatctccaattcgtaggtcaaaatac Has a 5′ G for 54Gguide 1 (SEQ ID NO: 641) U6 expression endogenous KRASGcgccaccagctccaacCaccacaagtttatattcagtcattttcagcagg Has a 5′ G for 54Fguide 1 (SEQ ID NO: 642) U6 expression endogenous KRASGtttctccatcaattacCacttgcttcctgtaggaatcctctattGTtgga Has a 5′ G for 54Fguide 3 (SEQ ID NO: 643) U6 expression endogenous KRASGctttctctcctgtagcCaaggccacaaaattatccactgtttttggaaca Has a 5′ G for 54Fguide 2 (SEQ ID NO: 644) U6 expression endogenous non-GTAATGCCTGGCTTGTCGACGCATAGTCTG Has a 5′ G for 54F targeting guide(SEQ ID NO: 645) U6 expression BoxB Cluc guidetctttccataGGCCCTGAAAAAGGGCCtgttctaaacCatcctgcggcctc Has a 5′ G for 62BtactcGGCCCTGAAAAAGGGCCattcaattac U6 expression (SEQ ID NO: 646)BoxB non- cagctggcgaGGCCCTGAAAAAGGGCCggggatgtgcCgcaaggcgattaaHas a 5′ G for 62B targeting guide gttggGGCCCTGAAAAAGGGCCacgccagggtU6 expression (SEQ ID NO: 647) Stafforst fullGTGGAATAGTATAACAATATGCTAAATGTTGTTATAGTATCCCACtctaaa Has a 5′ G for 62Clength ADAR2 CCAtcctgcgGGGCCCTCTTCAGGGCCC U6 expression guide 1(SEQ ID NO: 648) Stafforst fullGTGGAATAGTATAACAATATGCTAAATGTTGTTATAGTATCCCACaccctg Has a 5′ G for 62Clength ADAR2 gcgttacccaGGGCCCTCTTCAGGGCCC U6 expression non-targeting(SEQ ID NO: 649) guide

REFERENCES

-   1. P. D. Hsu, E. S. Lander, F. Zhang, Development and applications    of CRISPR-Cas9 for genome engineering. Cell 157, 1262-1278 (2014).-   2. A. C. Komor, A. H. Badran, D. R. Liu, CRISPR-Based Technologies    for the Manipulation of Eukaryotic Genomes. Cell 168, 20-36 (2017).-   3. L. Cong et al., Multiplex genome engineering using CRISPR/Cas    systems. Science 339, 819-823 (2013).-   4. P. Mali et al., RNA-guided human genome engineering via Cas9.    Science 339, 823-826 (2013).-   5. B. Zetsche et al., Cpf1 is a single RNA-guided endonuclease of a    class 2 CRISPR-Cas system. Cell 163, 759-771 (2015).-   6. H. Kim, J. S. Kim, A guide to genome engineering with    programmable nucleases. Nat Rev Genet 15, 321-334 (2014).-   7. A. C. Komor, Y. B. Kim, M. S. Packer, J. A. Zuris, D. R. Liu,    Programmable editing of a target base in genomic DNA without    double-stranded DNA cleavage. Nature 533, 420-424 (2016).-   8. K. Nishida et al., Targeted nucleotide editing using hybrid    prokaryotic and vertebrate adaptive immune systems. Science 353,    (2016).-   9. Y. B. Kim et al., Increasing the genome-targeting scope and    precision of base editing with engineered Cas9-cytidine deaminase    fusions. Nat Biotechnol 35, 371-376 (2017).-   10. O. O. Abudayyeh et al., C2c2 is a single-component programmable    RNA-guided RNA-targeting CRISPR effector. Science 353, aaf5573    (2016).-   11. S. Shmakov et al., Discovery and Functional Characterization of    Diverse Class 2 CRISPR-Cas Systems. Mol Cell 60, 385-397 (2015).-   12. S. Shmakov et al., Diversity and evolution of class 2 CRISPR-Cas    systems. Nat Rev Microbiol 15, 169-182 (2017).-   13. A. A. Smargon et al., Cas13b Is a Type VI-B CRISPR-Associated    RNA-Guided RNase Differentially Regulated by Accessory Proteins    Csx27 and Csx28. Mol Cell 65, 618-630 e617 (2017).-   14. J. S. Gootenberg et al., Nucleic acid detection with    CRISPR-Cas13a/C2c2. Science 356, 438-442 (2017).-   15. O. O. Abudayyeh et al., RNA targeting with CRISPR-Cas13a. Nature    in press, (2017).-   16. K. Nishikura, Functions and regulation of RNA editing by ADAR    deaminases. Annu Rev Biochem 79, 321-349 (2010).-   17. B. L. Bass, H. Weintraub, An unwinding activity that covalently    modifies its double-stranded RNA substrate. Cell 55, 1089-1098    (1988).-   18. M. M. Matthews et al., Structures of human ADAR2 bound to dsRNA    reveal base-flipping mechanism and basis for site selectivity. Nat    Struct Mol Biol 23, 426-433 (2016).-   19. A. Kuttan, B. L. Bass, Mechanistic insights into editing-site    specificity of ADARs. Proc Natl Acad Sci U S A 109, E3295-3304    (2012).-   20. S. K. Wong, S. Sato, D. W. Lazinski, Substrate recognition by    ADAR1 and ADAR2. RNA 7, 846-858 (2001).-   21. M. Fukuda et al., Construction of a guide-RNA for site-directed    RNA mutagenesis utilising intracellular A-to-I RNA editing. Sci Rep    7, 41478 (2017).-   22. M. F. Montiel-Gonzalez, I. Vallecillo-Viejo, G. A.    Yudowski, J. J. Rosenthal, Correction of mutations within the cystic    fibrosis transmembrane conductance regulator by site-directed RNA    editing. Proc Natl Acad Sci U S A 110, 18285-18290 (2013).-   23. M. F. Montiel-Gonzalez, I. C. Vallecillo-Viejo, J. J. Rosenthal,    An efficient system for selectively altering genetic information    within mRNAs. Nucleic Acids Res 44, e157 (2016).-   24. J. Wettengel, P. Reautschnig, S. Geisler, P. J. Kahle, T.    Stafforst, Harnessing human ADAR2 for RNA repair—Recoding a PINK1    mutation rescues mitophagy. Nucleic Acids Res 45, 2797-2808 (2017).-   25. Y. Wang, J. Havel, P. A. Beal, A Phenotypic Screen for    Functional Mutants of Human Adenosine Deaminase Acting on RNA 1. ACS    Chem Biol 10, 2512-2519 (2015).-   26. K. A. Lehmann, B. L. Bass, Double-stranded RNA adenosine    deaminases ADAR1 and ADAR2 have overlapping specificities.    Biochemistry 39, 12875-12884 (2000).-   27. Y. Zheng, C. Lorenzo, P. A. Beal, DNA editing in DNA/RNA hybrids    by adenosine deaminases that act on RNA. Nucleic Acids Res 45,    3369-3377 (2017).-   28. K. Gao et al., A de novo loss-of-function GRIN2A mutation    associated with childhood focal epilepsy and acquired epileptic    aphasia. PLoS One 12, e0170818 (2017).-   29. H. M. Lanoiselee et al APP, PSEN1, and PSEN2 mutations in    early-onset Alzheimer disease: A genetic screening study of familial    and sporadic cases. PLoS Med 14, e1002270 (2017).-   30. C. Ballatore, V. M. Lee, J. Q. Trojanowski, Tau-mediated    neurodegeneration in Alzheimer's disease and related disorders. Nat    Rev Neurosci 8, 663-672 (2007).-   31. Y. Li et al., Carriers of rare missense variants in IFIH1 are    protected from psoriasis. J Invest Dermatol 130, 2768-2772 (2010).-   32. R. S. Finkel et al., Treatment of infantile-onset spinal    muscular atrophy with nusinersen: a phase 2, open-label,    dose-escalation study. Lancet 388, 3017-3026 (2016).

Example 4

Truncations of dCas13b were generated for RNA editing.

Truncations from both N and C terminal ends in 20 amino acid intervalswere generated and tested in context of A-to-I RNA editing.dCas13b6(Δ795-1095)-GS-HIVNES-GS-huADAR2dd was selected as the finalconstruct for packaging into AAV.

Cas13b orthologs including Cas13b6 (RanCas13b), Cas13b11 (PguCas13b),and Cas13b12 (PspCas13b) were tested. For each ortholog, weprogressively truncated off 20 amino acids from each of the N terminal(red) and C terminal (blue) ends of the Cas13b ortholog. We then fusedthis truncated protein to ADAR2dd(E488Q) and evaluated the fusionprotein for RNA editing activity of a premature stop codon in Cypridinaluciferase, with Gaussia luciferase as an expression control. We foundthat truncating 300 amino acids off the C-terminal end of Cas13b6(RanCas13b) allowed for retention of about ⅔ of RNA editing activitywhile shortening the protein to be acceptably close to the packaginglimit of AAV. AAV is generally able to package genomes of about 4.7 kb,with packaging efficiency decreasing for larger genomes. The testresults on Cas13b6, Cas13b11, and Cas13b12 are shown in FIGS. 67-69,respectively.

The dCas13b6(Δ795-1095)-REPAIR construct was delivered to primary neuroncultures using AAV. Primary rat cortical neuron culture was used fortesting editing of MAP2 transcript. For delivery, AAV1/AAV2 hybridcapsids were used. Mismatch distance, which was distance from directrepeat to mismatched base in guide RNA spacer sequence, was measured.

We used Cas13b6 with 300 amino acids removed from the C terminal endfused to ADAR2dd(E488Q) as in the schematic in FIG. 70 to make a silentmutation to the MAP2 transcript in primary rat cortical neurons. MAP2 isa neuronal-specific marker, and additionally, the REPAIR protein wasexpressed under a human synapsin promoter to increase neuron-specificexpression of the system. We delivered this to primary rat corticalneuron cultures using AAV1/AAV2 hybrid capsids, and achieved up to 20%editing for specified guide RNA designs at the target site (FIG. 70).

We additionally used the system to modulate editing at a site in thepotassium ion channel Kcna1 that is already a natural ADAR2 substrate bydelivering the system detailed on FIG. 71 using AAV1/2 hybrid capsids inprimary rat cortical neuron culture. We found that, while we were ableto increase the percent of edited transcripts by delivering our REPAIRsystem to these cells, a guide RNA was not necessarily needed to directthis activity. This may be because this is already a natural substrateof ADAR2, so a guideRNA was not necessary to induce editing at thissite. The result suggests that overexpressing our construct, whichcontained the ADAR2 catalytic domain, was likely enough to startsaturating editing at these natural substrates.

FIG. 72 shows a schematic of a plasmid containing thedCas13b6(Δ795-1095)-GS-HIVNES-GS-huADAR2dd. The sequence of the plasmidis shown in the table below.

TABLE 22 Sequence of the plasmid shown in FIG. 72gacggatcgggagatctcccgatcccctatggtgcactctcagtacaatctgctctgatgccgcatagttaagccagtatctgctccctgcttgtgtgttggaggtcgctgagtagtgcgcgagcaaaatttaagctacaacaaggcaaggcttgaccgacaattgcatgaagaatctgcttagggttaggcgttttgcgctgcttcgcgatgtacgggccagatatacgcgttgacattgattattgactagttattaatagtaatcaattacggggtcattagttcatagcccatatatggagttccgcgttacataacttacggtaaatggcccgcctggctgaccgcccaacgacccccgcccattgacgtcaataatgacgtatgttcccatagtaacgccaatagggactttccattgacgtcaatgggtggagtatttacggtaaactgcccacttggcagtacatcaagtgtatcatatgccaagtacgccccctattgacgtcaatgacggtaaatggcccgcctggcattatgcccagtacatgaccttatgggactttcctacttggcagtacatctacgtattagtcatcgctattaccatggtgatgcggttttggcagtacatcaatgggcgtggatagcggtttgactcacggggatttccaagtctccaccccattgacgtcaatgggagtttgttttggcaccaaaatcaacgggactttccaaaatgtcgtaacaactccgccccattgacgcaaatgggcggtaggcgtgtacggtgggaggtctatataagcagagctctctggctaactagagaacccactgcttactggcttatcgaaattaatacgactcactatagggagacccaagctggcagcgtttaaacttaagcttgccaccATGgagaagcccctgctgcctaacgtgtataccctgaagcacaagttcttttggggcgccttcctgaatatcgcccggcacaacgcctttatcaccatctgccacatcaatgagcagctgggcctgaaaacccccagcaacgacgataagatcgtggacgtggtgtgcgagacatggaacaatatcctgaacaatgaccacgatctgctgaagaagtcccagctgaccgagctgatcctgaagcacttcccttttctgacagccatgtgctaccacccccctaagaaggagggcaagaagaagggccaccagaaggagcagcagaaggagaaggagagcgaggcacagtcccaggcagaggccctgaacccatccaagctgatcgaggccctggagatcctggtgaatcagctgcactctctgGCAaactactatagcGCCtataagcacaagaagcccgacgccgagaaggatatcttcaagcacctgtataaggccttcgacgcctctctgagaatggtgaaggaggattataaggcccacttcaccgtgaatctgacaagggactttgcccacctgaaccgcaagggcaagaacaagcaggacaatcccgacttcaacagatacagattcgagaaggatggcttctttaccgagtctggcctgctgttctttacaaatctgtttctggacaagagggatgcctattggatgctgaagaaggtgtccggcttcaaggcctctcacaagcagcgcgagaagatgaccacagaggtgttttgcaggagccgcatcctgctgcccaagctgaggctggagtcccgctacgaccacaaccagatgctgctggatatgctgtctgagctgagcagatgtcctaagctgctgtatgagaagctgagcgaggagaataagaagcacttccaggtggaggccgacggctttctggatgagatcgaggaggagcagaacccattcaaggacaccctgatccggcaccaggatagattcccctactttgccctgaggtatctggacctgaatgagtccttcaagtctatccgctttcaggtggatctgggcacataccactattgtatctacgacaagaagatcggcgatgagcaggagaagaggcacctgacccgcacactgctgtccttcggccggctgcaggactttaccgagatcaacagaccccaggagtggaaggccctgaccaaggacctggattacaaggagacatccaatcagcctttcatctctaagaccacaccacactatcacatcaccgacaacaagatcggctttcggctgggcacaagcaaggagctgtacccctccctggagatcaaggatggcgccaatagaatcgccaagtacccttataactccggcttcgtggcccacgcctttatctctgtgcacgagctgctgcctctgatgttctaccagcacctgaccggcaagagcgaggacctgctgaaggagacagtgcggcacatccagagaatctataaggacttcgaggaggagcggatcaataccatcgaggatctggagaaggcaaaccagggcagactgccactgggagcctttcccaagcagatgctgggcctgctgcagaataagcagcctgatctgtccgagaaggccaagatcaagatcgagaagctgatcgccgagacaaagctgctgtctcacaggctgaacacaaagctgaagagctccccaaagctgggcaagcggagagagaagctgatcaagacaggcgtgctggccgactggctggtgaaggatttcatgcgctttcagcccgtggcctacgacgcccagaatcagcctatcaagtctagcaaggccaactccaccgagttctggtttatcaggcgcgccctggccctgtacggaggagagaagaataggctggagggctatttcaagcagaccaacctgatcggcaacacaaatccacaccccttcctgaacaagtttaattggaaggcctgcaggaatctggtggatttctaccagcagtatctggagcagcgcgagaagtttctggaggccatcaagaaccagccatgggagccctaccagtattgcctctgctgaagatccctaaggagaacaggaagaatctggtgaagggatgggagcagggaggaatctctctgccacggggcctgtttaccgaggccatcagagagacactgtctgaggacctgatgctgagcaagccaatccgcaaggagatcaagaagcacggccgggtgggcttcatcagcagagccatcaccctgtactttaaggagaagtatcaggataagcaccagagcttctacaatctgtcctataagctggaggcaaaggcaccactgggatcTCTTCAACTGCCTCCACTTGAAAGACTGACACTGGGATCCcagctgcatttaccgcaggttttagctgacgctgtctcacgcctggtcctgggtaagtttggtgacctgaccgacaacttctcctcccctcacgctcgcagaaaagtgctggctggagtcgtcatgacaacaggcacagatgttaaagatgccaaggtgataagtgtttctacaggaacaaaatgtattaatggtgaatacatgagtgatcgtggccttgcattaaatgactgccatgcagaaataatatctcggagatccttgctcagatttctttatacacaacttgagctttacttaaataacaaagatgatcaaaaaagatccatctttcagaaatcagagcgaggggggtttaggctgaaggagaatgtccagtttcatctgtacatcagcacctctccctgtggagatgccagaatcttctcaccacatgagccaatcctggaagaaccagcagatagacacccaaatcgtaaagcaagaggacagctacggaccaaaatagagtctggtCaggggacgattccagtgcgctccaatgcgagcatccaaacgtgggacggggtgctgcaaggggagcggctgctcaccatgtcctgcagtgacaagattgcacgctggaacgtggtgggcatccagggatcActgctcagcattttcgtggagcccatttacttctcgagcatcatcctgggcagcctttaccacggggaccacctttccagggccatgtaccagcggatctccaacatagaggacctgccacctctctacaccctcaacaagcctttgctcagtggcatcagcaatgcagaagcacggcagccagggaaggcccccaacttcagtgtcaactggacggtaggcgactccgctattgaggtcatcaacgccacgactgggaaggatgagctgggccgcgcgtcccgcctgtgtaagcacgcgttgtactgtcgctggatgcgtgtgcacggcaaggttccctcccacttactacgctccaagattaccaagcccaacgtgtaccatgagtccaagctggcggcaaaggagtaccaggccgccaaggcgcgtctgttcacagccttcatcaaggcggggctgggggcctgggtggagaagcccaccgagcaggaccagttctcactcacgTAAgcggccgctcgagtctagagggcccgtttaaacccgctgatcagcctcgactgtgccttctagttgccagccatctgttgtttgcccctcccccgtgccttccttgaccctggaaggtgccactcccactgtcctttcctaataaaatgaggaaattgcatcgcattgtctgagtaggtgtcattctattctggggggtggggtggggcaggacagcaagggggaggattgggaagacaatagcaggcatgctggggatgcggtgggctctatggcttctgaggcggaaagaaccagctggggctctagggggtatccccacgcgccctgtagcggcgcattaagcgcggcgggtgtggtggttacgcgcagcgtgaccgctacacttgccagcgccctagcgcccgctcctttcgctttcttcccttcctttctcgccacgttcgccggctttccccgtcaagctctaaatcgggggctccctttagggttccgatttagtgctttacggcacctcgaccccaaaaaacttgattagggtgatggttcacgtagtgggccatcgccctgatagacggtttttcgccctttgacgttggagtccacgttctttaatagtggactcttgttccaaactggaacaacactcaaccctatctcggtctattcttttgatttataagggattttgccgatttcggcctattggttaaaaaatgagctgatttaacaaaaatttaacgcgaattaattctgtggaatgtgtgtcagttagggtgtggaaagtccccaggctccccagcaggcagaagtatgcaaagcatgcatctcaattagtcagcaaccaggtgtggaaagtccccaggctccccagcaggcagaagtatgcaaagcatgcatctcaattagtcagcaaccatagtcccgcccctaactccgcccatcccgcccctaactccgcccagttccgcccattctccgccccatggctgactaattttttttatttatgcagaggccgaggccgcctctgcctctgagctattccagaagtagtgaggaggcttttttggaggcctaggcttttgcaaaaagctcccgggagcttgtatatccattttcggatctgatcaagagacaggatgaggatcgtttcgcatgattgaacaagatggattgcacgcaggttctccggccgcttgggtggagaggctattcggctatgactgggcacaacagacaatcggctgctctgatgccgccgtgttccggctgtcagcgcaggggcgcccggttctttttgtcaagaccgacctgtccggtgccctgaatgaactgcaggacgaggcagcgcggctatcgtggctggccacgacgggcgttccttgcgcagctgtgctcgacgttgtcactgaagcgggaagggactggctgctattgggcgaagtgccggggcaggatctcctgtcatctcaccttgctcctgccgagaaagtatccatcatggctgatgcaatgcggcggctgcatacgcttgatccggctacctgcccattcgaccaccaagcgaaacatcgcatcgagcgagcacgtactcggatggaagccggtcttgtcgatcaggatgatctggacgaagagcatcaggggctcgcgccagccgaactgttcgccaggctcaaggcgcgcatgcccgacggcgaggatctcgtcgtgacccatggcgatgcctgcttgccgaatatcatggtggaaaatggccgcttttctggattcatcgactgtggccggctgggtgtggcggaccgctatcaggacatagcgttggctacccgtgatattgctgaagagcttggcggcgaatgggctgaccgcttcctcgtgctttacggtatcgccgctcccgattcgcagcgcatcgccttctatcgccttcttgacgagttcttctgagcgggactctggggttcgaaatgaccgaccaagcgacgcccaacctgccatcacgagatttcgattccaccgccgccttctatgaaaggttgggcttcggaatcgttttccgggacgccggctggatgatcctccagcgcggggatctcatgctggagttcttcgcccaccccaacttgtttattgcagcttataatggttacaaataaagcaatagcatcacaaatttcacaaataaagcatttttttcactgcattctagttgtggtttgtccaaactcatcaatgtatcttatcatgtctgtataccgtcgacctctagctagagcttggcgtaatcatggtcatagctgtttcctgtgtgaaattgttatccgctcacaattccacacaacatacgagccggaagcataaagtgtaaagcctggggtgcctaatgagtgagctaactcacattaattgcgttgcgctcactgcccgctttccagtcgggaaacctgtcgtgccagctgcattaatgaatcggccaacgcgcggggagaggcggtttgcgtattgggcgctcttccgcttcctcgctcactgactcgctgcgctcggtcgttcggctgcggcgagcggtatcagctcactcaaaggcggtaatacggttatccacagaatcaggggataacgcaggaaagaacatgtgagcaaaaggccagcaaaaggccaggaaccgtaaaaaggccgcgttgctggcgtttttccataggctccgcccccctgacgagcatcacaaaaatcgacgctcaagtcagaggtggcgaaacccgacaggactataaagataccaggcgtttccccctggaagctccctcgtgcgctctcctgttccgaccctgccgcttaccggatacctgtccgcctttctcccttcgggaagcgtggcgctttctcatagctcacgctgtaggtatctcagttcggtgtaggtcgttcgctccaagctgggctgtgtgcacgaaccccccgttcagcccgaccgctgcgccttatccggtaactatcgtcttgagtccaacccggtaagacacgacttatcgccactggcagcagccactggtaacaggattagcagagcgaggtatgtaggcggtgctacagagttcttgaagtggtggcctaactacggctacactagaagaacagtatttggtatctgcgctctgctgaagccagttaccttcggaaaaagagttggtagctcttgatccggcaaacaaaccaccgctggtagcggtttttttgtttgcaagcagcagattacgcgcagaaaaaaaggatctcaagaagatcctttgatcttttctacggggtctgacgctcagtggaacgaaaactcacgttaagggattttggtcatgagattatcaaaaaggatcttcacctagatccttttaaattaaaaatgaagttttaaatcaatctaaagtatatatgagtaaacttggtctgacagttaccaatgcttaatcagtgaggcacctatctcagcgatctgtctatttcgttcatccatagttgcctgactccccgtcgtgtagataactacgatacgggagggcttaccatctggccccagtgctgcaatgataccgcgagacccacgctcaccggctccagatttatcagcaataaaccagccagccggaagggccgagcgcagaagtggtcctgcaactttatccgcctccatccagtctattaattgttgccgggaagctagagtaagtagttcgccagttaatagtttgcgcaacgttgttgccattgctacaggcatcgtggtgtcacgctcgtcgtttggtatggcttcattcagctccggttcccaacgatcaaggcgagttacatgatcccccatgttgtgcaaaaaagcggttagctccttcggtcctccgatcgttgtcagaagtaagttggccgcagtgttatcactcatggttatggcagcactgcataattctcttactgtcatgccatccgtaagatgcttttctgtgactggtgagtactcaaccaagtcattctgagaatagtgtatgcggcgaccgagttgctcttgcccggcgtcaatacgggataataccgcgccacatagcagaactttaaaagtgctcatcattggaaaacgttcttcggggcgaaaactctcaaggatcttaccgctgttgagatccagttcgatgtaacccactcgtgcacccaactgatcttcagcatcttttactttcaccagcgtttctgggtgagcaaaaacaggaaggcaaaatgccgcaaaaaagggaataagggcgacacggaaatgttgaatactcatactcttcctttttcaatattattgaagcatttatcagggttattgtctcatgagcggatacatatttgaatgtatttagaaaaataaacaaataggggttccgcgcacatttccccgaaaagtgccacctgacgtc

Example 5

Design and Cloning of Mammalian Constructs for RNA Editing

To generate truncated versions of Cas12b, primers were designed to PCRamplify the dCas13b that truncated off 60 bp (20 amino acids)progressively up to 900 bp off of the C terminal end (15 truncations intotal), and these truncated Cas13b genes were Gibson cloned into thepcDNA-CMV-ADAR2 backbone described above. Guide RNAs targeting Cluc werecloned using golden gate cloning into a mammalian expression vectorcontaining the direct repeat sequence for this ortholog at the 3′ end ofthe spacer sequence destination site, under the U6 promoter.

The luciferase reporter used was a CMV-Cluc (W85X) EF1alpha-Gluc dualluciferase reporter used by Cox et. al. (2017) to measure RNA editing.This reporter vector expresses functional Gluc as a normalizationcontrol, but a defective Cluc due to the addition of the W85Xpretermination site.

REPAIR Editing in Mammalian Cells

To assess REPAIR activity in mammalian cells, we transfected 150 ng ofREPAIR vector, 300 ng of guide expression plasmid, and 45 ng of the RNAediting reporter. We then harvested media with the secreted luciferaseafter 48 hours and diluted the media 1:10 in Dulbecco's phosphatebuffered saline (PBS) (10 μl) of media into 90 μl PBS). We measuredluciferase activity with BioLux Cypridinia and Biolux Gaussia luciferaseassay kits (New England Biolabs) on a plate reader (Biotek Synergy Neo2)with an injection protocol. All replicates performed are biologicalreplicates.

Nuclease-dead Cas13b fused to an ADAR deaminase domain was used forREPAIR to achieve targeted RNA base editing. AAV-mediated delivery iscommonly used for gene therapies, but REPAIR exceeded the size limit ofAAV's cargo capacity. We showed previously that C-terminal truncationsof Prevotella sp. P5-125 (PspCas13b) did not decrease REPAIR activity.We further used another ortholog of Cas13b, from Porphyromonas gulae(PguCas13b), which was stably expressed and showed high activity inmammalian cells, in contrast to PbuCas13b. Based on alignments betweenPbuCas13b and PguCas13b, we made PguCas13b truncations to remove theHEPN2 domain, fused it to ADAR, and tested its ability to carry out baseediting with the REPAIR system. Surprisingly, not only did thesetruncated mutants retain RNA targeting, some were significantly moreefficient at RNA editing (FIG. 73).

Various modifications and variations of the described methods,pharmaceutical compositions, and kits of the invention will be apparentto those skilled in the art without departing from the scope and spiritof the invention. Although the invention has been described inconnection with specific embodiments, it will be understood that it iscapable of further modifications and that the invention as claimedshould not be unduly limited to such specific embodiments. Indeed,various modifications of the described modes for carrying out theinvention that are obvious to those skilled in the art are intended tobe within the scope of the invention. This application is intended tocover any variations, uses, or adaptations of the invention following,in general, the principles of the invention and including suchdepartures from the present disclosure come within known customarypractice within the art to which the invention pertains and may beapplied to the essential features herein before set forth.

1. (canceled)
 2. (canceled)
 3. An engineered, non-naturally occurringsystem comprising a catalytically inactive Cas13 effector protein(dCas13) or a nucleotide sequence encoding the catalytically inactiveCas13 effector protein, wherein the dCas13 protein is truncated at a Cterminus, an N terminus, or both, and wherein the dCas13 is truncated byat least 20, at least 40, at least 60, at least 80, at least 100, atleast 120, at least 140, at least 160, at least 180, at least 200, atleast 220, at least 240, at least 260, or at least 300 amino acids onthe C terminus, wherein the Cas13 effector protein is Cas13a, andwherein the truncated form of the Cas13 effector protein has beentruncated at C-terminal Δ984-1090.
 4. (canceled)
 5. An engineered,non-naturally occurring system comprising a catalytically inactive Cas13effector protein (dCas13) or a nucleotide sequence encoding thecatalytically inactive Cas13 effector protein, wherein the dCas13protein is truncated at a C terminus, an N terminus, or both, whereinthe Cas13 effector protein is Cas13a, and wherein the truncated form ofthe Cas13 effector protein has been truncated at C-terminal Δ984-1090,wherein amino acid positions of the truncations correspond to amino acidpositions of Prevotella sp. P5-125 Cas13b protein.
 6. (canceled) 7.(canceled)
 8. (canceled)
 9. (canceled)
 10. (canceled)
 11. (canceled) 12.(canceled)
 13. (canceled)
 14. (canceled)
 15. (canceled)
 16. (canceled)17. (canceled)
 18. An engineered, non-naturally occurring systemcomprising a catalytically inactive Cas13 effector protein (dCas13) or anucleotide sequence encoding the catalytically inactive Cas13 effectorprotein, wherein the Cas13 effector protein is Cas13a, wherein thesystem further comprises a functional component or wherein thenucleotide sequence further encodes the functional component, whereinthe functional component is a base editing component, wherein the baseediting component comprises an adenosine deaminase or a catalytic domainthereof, and wherein the adenosine deaminase or the catalytic domainthereof, is inserted into an internal loop of the dCas13.
 19. (canceled)20. (canceled)
 21. (canceled)
 22. (canceled)
 23. (canceled) 24.(canceled)
 25. (canceled)
 26. (canceled)
 27. (canceled)
 28. (canceled)29. (canceled)
 30. (canceled)
 31. (canceled)
 32. (canceled) 33.(canceled)
 34. (canceled)
 35. A vector system comprising one or morevectors encoding the dCas13 of claim
 3. 36. A vector system comprisingone or more vectors encoding the dCas13 of claim
 5. 37. A vector systemcomprising one or more vectors encoding the dCas13 of claim
 18. 38. Anin vitro or ex vivo host cell or progeny thereof or cell line or progenythereof comprising the system of claim
 3. 39. An in vitro or ex vivohost cell or progeny thereof or cell line or progeny thereof comprisingthe system of claim
 5. 40. An in vitro or ex vivo host cell or progenythereof or cell line or progeny thereof comprising the system of claim18.
 41. A method for programmable and targeted base editing of a targetsequence comprising delivering of the system of claim 3 to a cell. 42.The method of claim 41, further comprising determining the targetsequence of interest and selecting an adenosine deaminase or catalyticdomain thereof which most efficiently deaminates an adenine present inthe target sequence.
 43. A method for programmable and targeted baseediting of a target sequence comprising delivering of the system ofclaim 5 to a cell.
 44. The method of claim 43, further comprisingdetermining the target sequence of interest and selecting an adenosinedeaminase or catalytic domain thereof which most efficiently deaminatesan adenine present in the target sequence.
 45. A method for programmableand targeted base editing of a target sequence comprising delivering ofthe system of claim 18 to a cell.
 46. The method of claim 45, furthercomprising determining the target sequence of interest and selecting anadenosine deaminase or catalytic domain thereof which most efficientlydeaminates an adenine present in the target sequence.
 47. A method forprogrammable and targeted base editing of a target sequence comprisingdelivering an engineered, non-naturally occurring system comprising acatalytically inactive Cas13 effector protein (dCas13) or a nucleotidesequence encoding the catalytically inactive Cas13 effector protein,wherein the Cas13 effector protein is Cas13a, wherein the Cas13 effectorprotein has been truncated at C-terminal Δ984-1090, to a cell, whereinthe method further comprises determining the target sequence of interestand selecting an adenosine deaminase or catalytic domain thereof whichmost efficiently deaminates an adenine present in the target sequence,and wherein the deamination remedies a disease caused by transcriptscontaining a pathogenic G→A or C→T point mutation.
 48. The method ofclaim 47, wherein the dCas13 is truncated by at least 20, at least 40,at least 60, at least 80, at least 100, at least 120, at least 140, atleast 160, at least 180, at least 200, at least 220, at least 240, atleast 260, or at least 300 amino acids on the N terminus.
 49. The methodof claim 47, wherein the dCas13 comprises a truncated form of a Cas13effector protein at an HEPN domain of the Cas13 effector protein. 50.The method of claim 47, further comprising a functional component orwherein the nucleotide sequence further encodes the functionalcomponent.
 51. The method of claim 50, wherein the functional componentis a base editing component.
 52. The method of claim 51, wherein thebase editing component comprises an adenosine deaminase or a catalyticdomain thereof.
 53. The method of claim 52, wherein the adenosinedeaminase or the catalytic domain thereof, is fused to the dCas13. 54.The method of claim 53, wherein the adenosine deaminase or the catalyticdomain thereof, is fused to the dCas13 by a linker.
 55. The method ofclaim 52, wherein the adenosine deaminase or the catalytic domainthereof, is inserted into an internal loop of the dCas13.
 56. The methodof claim 52, wherein the adenosine deaminase or the catalytic domainthereof, is linked to an adaptor protein.
 57. The method of claim 56,wherein the adaptor protein is MS2.
 58. The method of claim 52, whereinthe adenosine deaminase or the catalytic domain thereof, is a human,cephalopod, or Drosophila protein.